WO1999049029A1 - Control of gene expression - Google Patents

Control of gene expression Download PDF

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Publication number
WO1999049029A1
WO1999049029A1 PCT/AU1999/000195 AU9900195W WO9949029A1 WO 1999049029 A1 WO1999049029 A1 WO 1999049029A1 AU 9900195 W AU9900195 W AU 9900195W WO 9949029 A1 WO9949029 A1 WO 9949029A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
plasmid
gene
cell
pvy
Prior art date
Application number
PCT/AU1999/000195
Other languages
French (fr)
Inventor
Michael Wayne Graham
Robert Norman Rice
Original Assignee
Benitec Australia Ltd
State Of Queensland Through Its Department Of Primary Industries
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=25645735&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO1999049029(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority claimed from AUPP2499A external-priority patent/AUPP249998A0/en
Priority claimed from AUPP2492A external-priority patent/AUPP249298A0/en
Priority to EP10183258.2A priority Critical patent/EP2302057B1/en
Priority to EP99910039A priority patent/EP1071762A4/en
Priority to PL99343064A priority patent/PL343064A1/en
Priority to KR1020107006892A priority patent/KR101054060B1/en
Priority to GB0024727A priority patent/GB2353282C/en
Priority to BRPI9908967A priority patent/BRPI9908967B1/en
Priority to SG200503921-9A priority patent/SG141233A1/en
Priority to CA002323726A priority patent/CA2323726C/en
Priority to KR1020007010419A priority patent/KR20010042069A/en
Application filed by Benitec Australia Ltd, State Of Queensland Through Its Department Of Primary Industries filed Critical Benitec Australia Ltd
Priority to AU29163/99A priority patent/AU743316C/en
Priority to SG200205122A priority patent/SG115493A1/en
Priority to SK1372-2000A priority patent/SK287538B6/en
Priority to NZ506648A priority patent/NZ506648A/en
Priority to EP07008204.5A priority patent/EP1857549B1/en
Priority to KR1020067005341A priority patent/KR101085210B1/en
Priority to JP2000537990A priority patent/JP4187413B2/en
Priority to HU0101225A priority patent/HU230353B1/en
Publication of WO1999049029A1 publication Critical patent/WO1999049029A1/en
Priority to HK01105904A priority patent/HK1035742A1/en
Priority to US10/646,070 priority patent/US7754697B2/en
Priority to US11/218,999 priority patent/US8168774B2/en
Priority to US13/458,704 priority patent/US20120277285A1/en
Priority to US14/137,737 priority patent/US9963698B2/en

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    • C12N2840/00Vectors comprising a special translation-regulating system
    • C12N2840/20Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • the present invention relates generally to a method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention utilises recombinant DNA technology to post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, organ or whole organism, thereby producing novel phenotypes. Novel synthetic genes and genetic constructs which are capable of repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.
  • derived from shall be taken to indicate that a specified integer may be obtained from a particular specified source or species, albeit not necessarily directly from that specified source or species.
  • Sequence identity numbers (SEQ ID NOS.) containing nucleotide and amino acid sequence information included in this specification are collected after the Abstract and have been prepared using the programme Patentln Version 2.0. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (e.g. ⁇ 210>1 , ⁇ 210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields ⁇ 211 >, ⁇ 212> and ⁇ 213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field ⁇ 400> followed by the sequence identifier (eg. ⁇ 400>1 , ⁇ 400>2, etc).
  • nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
  • amino acid residues referred to herein, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission are listed in Table 1.
  • Controlling metabolic pathways in eukaryotic organisms is desirable for the purposes of producing novel traits therein or introducing novel traits into a particular cell, tissue or organ of said organism.
  • recombinant DNA technology has provided significant progress in an understanding of the mechanisms regulating eukaryotic gene expression, much less progress has been made in the actual manipulation of gene expression to produce novel traits.
  • human intervention may lead to a modulation of the level of eukaryotic gene expression.
  • mRNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed and capable of being translated into a polypeptide.
  • a double-stranded mRNA may form by base pairing between the complementary nucleotide sequences, to produce a complex which is translated at low efficiency and/or degraded by intracellular ribonuclease enzymes prior to being translated.
  • an endogenous gene in a cell, tissue or organ may be suppressed when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell.
  • this approach has not been established and appears to be involve mechanistically heterogeneous processes.
  • this approach has been postulated to involve transcriptional repression, in which case somatically-heritable repressed states of chromatin are formed or alternatively, a post-transcriptional silencing wherein transcription initiation occurs normally but the RNA products of the co-suppressed genes are subsequently eliminated.
  • transcriptional repression such as the Drosophila Pc-G system
  • Drosophila Pc-G system would appear to require some knowledge of the regulatory mechanisms capable of modulating the expression of any specific target gene and, as a consequence, would be difficult to implement in practice as a general technology for repressing, delaying or reducing gene expression in animal cells.
  • the invention is based in part on the surprising discovery by the inventors that cells which exhibit one or more desired traits can be produced and selected from transformed cells comprising a nucleic acid molecule operably linked to a promoter, wherein the transcription product of the nucleic acid molecule comprises a nucleotide sequence which is substantially identical to the nucleotide sequence of a transcript of an endogenous or non-endogenous target gene, the expression of which is intended to be modulated.
  • the transformed cells are regenerated into whole tissues, organs or organisms capable of exhibiting novel traits, in particular virus resistance and modified expression of endogenous genes.
  • one aspect of the present invention provides a method of modulating the expression of a target gene in an animal cell, tissue or organ, said method at least comprising the step of introducing to said cell, tissue or organ one or more dispersed nucleic acid molecules or foreign nucleic acid molecules comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or complementary thereto for a time and under conditions sufficient for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
  • the dispersed nucleic acid molecules or foreign nucleic acid molecules comprises a nucleotide sequence which encodes multiple copies of an mRNA molecule which is substantially identical to the nucleotide sequence of the mRNA product of the target gene. More preferably, the multiple copies of the target molecule are tandem direct repeat sequences.
  • the dispersed nucleic acid molecule or foreign nucleic acid molecule is in an expressible form such that it is at least capable of being transcribed to produce mRNA.
  • the target gene may be a gene which is endogenous to the animal cell or alternatively, a foreign gene such as a viral or foreign genetic sequence, amongst others.
  • a foreign gene such as a viral or foreign genetic sequence, amongst others.
  • the target gene is a viral genetic sequence.
  • the invention is particularly useful in the modulation of eukaryotic gene expression, in particular the modulation of human or animal gene expression and even more particularly in the modulation of expression of genes derived from vertebrate and invertebrate animals, such as insects, aquatic animals (eg. fish, shellfish, molluscs, crustaceans such as crabs, lobsters and prawns, avian animals and mammals, amongst others).
  • vertebrate and invertebrate animals such as insects, aquatic animals (eg. fish, shellfish, molluscs, crustaceans such as crabs, lobsters and prawns, avian animals and mammals, amongst others).
  • a variety of traits are selectable with appropriate procedures and sufficient numbers of transformed cells. Such traits include, but are not limited to, visible traits, disease- resistance traits, and pathogen-resistance traits.
  • the modulatory effect is applicable to a variety of genes expressed in plants and animals including, for example, endogenous genes responsible for cellular metabolism or cellular transformation, including oncogenes, transcription factors and other genes which encode polypeptides involved in cellular metabolism.
  • an alteration in the pigment production in mice can be engineered by - 8
  • tyrosinase gene therein. This provides a novel phenotype of albinism in black mice.
  • a genetic construct which comprises multiple copies of nucleotide sequence encoding a viral replicase, polymerase, coat protein or uncoating gene, or protease protein, may be introduced into a cell where it is expressed, to confer immunity against the virus upon the cell.
  • the dispersed nucleic acid molecule or foreign nucleic acid molecule will generally comprise a nucleotide sequence having greater than about 85% identity to the target gene sequence, however, a higher homology might produce a more effective modulation of expression of the target gene sequence. Substantially greater homology, or more than about 90% is preferred, and even more preferably about 95% to absolute identity is desirable.
  • the introduced dispersed nucleic acid molecule or foreign nucleic acid molecule sequence needing less than absolute homology, also need not be full length, relative to either the primary transcription product or fully processed mRNA of the target gene.
  • a higher homology in a shorter than full length sequence compensates for a longer less homologous sequence.
  • the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective. Normally, a sequence of greater than 20-100 nucleotides should be used, though a sequence of greater than about 200-300 nucleotides would be preferred, and a sequence of greater than 500-1000 nucleotides would be especially preferred depending on the size of the target gene.
  • a second aspect of the present invention provides a synthetic gene which is capable of modifying target gene expression in a cell, tissue or organ of a prokaryotic or eukaryotic organism which is transfected or transformed therewith, wherein said synthetic gene at least comprises a dispersed nucleic acid molecular foreign nucleic acid molecule comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a derivative thereof or a complementary sequence thereto placed operably under the control of a promoter sequence which is operable in said cell, tissue or organ.
  • a third aspect of the invention provides a synthetic gene which is capable of modifying the expression of a target gene in a cell, tissue or organ of a prokaryotic or eukaryotic organism which is transfected or transformed therewith, wherein said synthetic gene at least comprises multiple structural gene sequences, wherein each of said structural gene sequences comprises a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a derivative thereof or a complementary sequence thereto and wherein said multiple structural gene sequences are placed operably under the control of a single promoter sequence which is operable in said cell, tissue or organ.
  • a fourth aspect of the present invention provides a synthetic gene which is capable of modifying the expression of a target gene in a cell, tissue or organ of a prokaryote or eukaryote which is transfected or transformed therewith wherein said synthetic gene at least comprises multiple structural gene sequences wherein each of said structural gene sequences is placed operably under the control of a promoter sequence which is operable in said cell, tissue or organ and wherein each of said structural gene sequences comprises a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a derivative thereof or a complementary sequence thereto.
  • a fifth aspect of the present invention provides a genetic construct which is capable of modifying the expression of an endogenous gene or target gene in a transformed or transfected cell, tissue or organ wherein said genetic construct at least comprises the synthetic gene of the invention and one or more origins of replication and/or selectable marker gene sequences.
  • a sixth aspect of the invention provides a cell, tissue, organ or organism comprising the synthetic genes and genetic constructs described herein.
  • Figure 1 is a diagrammatic representation of the plasmid pEGFP-N1 MCS.
  • Figure 2 is a diagrammatic representation of the plasmid pCMV.cass.
  • Figure 3 is a diagrammatic representation of the plasmid pCMV.SV40L.cass.
  • Figure 4 is a diagrammatic representation of the plasmid pCMV.SV40LR.cass.
  • Figure 5 is a diagrammatic representation of the plasmid pCR.Bgl-GFP-Bam.
  • Figure 6 is a diagrammatic representation of the plasmid pBSII(SK+).EGFP.
  • Figure 7 is a diagrammatic representation of the plasmid pCMV.EGFP.
  • Figure 8 is a diagrammatic representation of the plasmid pCR.SV40L. - 11
  • Figure 9 is a diagrammatic representation of the plasmid pCR.BEVJ .
  • Figure 10 is a diagramma ic representation of the plasmid pCR.BEV.2.
  • Figure 11 is a diagramma' ic representation of the plasmid pCR.BEV.3.
  • Figure 12 is a diagramma ic representation of the plasmid pCMV.EGFP.BEV2.
  • Figure 13 is a diagramma ic representation of the plasmid pCMV.BEV.2.
  • Figure 14 is a diagramma ic representation of the plasmid pCMV.BEV.3.
  • Figure 15 is a diagramma ic representation of the plasmid pCMV.VEB.
  • Figure 16 is a diagramma ic representation of the plasmid pCMV.BEV.GFP.
  • Figure 17 is a diagramma ic representation of the plasmid pCMV.BEV.SV40L-0.
  • Figure 18 is a diagramma ic representation of the plasmid pCMV.0.SV40L.BEV.
  • Figure 19 is a diagramma ic representation of the plasmid pCMV.0.SV40L.VEB.
  • Figure 20 is a diagramma ic representation of the plasmid pCMV.BEVx2.
  • Figure 21 is a diagramma ic representation of the plasmid pCMV.BEVx3.
  • Figure 22 is a diagramma ic representation of the plasmid pCMV.BEVx4.
  • Figure 23 is a diagramma ic representation of the plasmid pCMV.BEV.SV40L.BEV. - 12 -
  • Figure 24 is a diagrammatic representation of the plasmid pCMV.BEV.SV40L.VEB.
  • Figure 25 is a diagrammatic representation of the plasmid pCMV.BEV.GFP.VEB.
  • Figure 26 is a diagrammatic representation of the plasmid pCMV.EGFP.BEV2.PFG.
  • Figure 27 is a diagrammatic representation of the plasmid pCMV.BEV.SV40LR.
  • Figure 28 is a diagrammatic representation of the plasmid pCDNA3.Galt.
  • Figure 29 is a diagrammatic representation of the plasmid pCMV.Galt.
  • Figure 30 is a diagrammatic representation of the plasmid pCMV.EGFP.Galt.
  • Figure 31 is a diagrammatic representation of the plasmid pCMV.Galt.GFP.
  • Figure 32 is a diagrammatic representation of the plasmid pCMV.Galt.SV40L.0.
  • Figure 33 is a diagrammatic representation of the plasmid pCMV.Galt.SV40L.tlaG.
  • Figure 34 is a diagrammatic representation of the plasmid pCMV.0.SV40L.Galt.
  • Figure 35 is a diagrammatic representation of the plasmid pCMV.Galtx2.
  • Figure 36 is a diagrammatic representation of the plasmid pCMV.Galtx4.
  • Figure 37 is a diagrammatic representation of the plasmid pCMV. Gait. SV40L. Gait.
  • Figure 38 is a diagrammatic representation of the plasmid pCMV.Galt. SV40L.tlaG. 13
  • Figure 39 is a diagramma ic representation of the plasmid pCMV.Galt.GFP.tlaG.
  • Figure 40 is a diagramma' ic representation of the plasmid pCMV.EGFP.Galt.PFG.
  • Figure 41 is a diagramma ic representation of the plasmid pCMV.Galt.SV40LR.
  • Figure 42 is a diagramma ic representation of the plasmid pART7.
  • Figure 43 is a diagramma c representation of the plasmid pART7.35S.SCBV.cass.
  • Figure 44 is a diagramma ic representation of the plasmid pBC.PVY.
  • Figure 45 is a diagramma ic representation of the plasmid pSP72.PVY.
  • Figure 46 is a diagramma ic representation of the plasmid pClapBC.PVY.
  • Figure 47 is a diagramma ic representation of the plasmid pBC.PVYx2.
  • Figure 48 is a diagramma ic representation of the plasmid pSP72.PVYx2.
  • Figure 49 is a diagramma ic representation of the plasmid pBC.PVYx3.
  • Figure 50 is a diagramma ic representation of the plasmid pBC.PVYx4.
  • Figure 51 is a diagramma ic representation of the plasmid pBC.PVY.LNYV.
  • Figure 52 is a diagramma' ic representation of the plasmid pBC.PVY.LNYV.PVY.
  • Figure 53 is a diagramma ic representation of the plasmid pBC.PVY.LNYV. YVP ⁇ . - 14 -
  • Figure 54 is a diagrammatic representation of the plasmid pBC.PVY.LNYV.YVP.
  • Figure 55 is a diagrammatic representation of the plasmid pART27.PVY
  • Figure 56 is a diagrammatic representation of the plasmid pART27.35S.PVY.SCBV.O.
  • Figure 57 is a diagrammatic representation of the plasmid pART27.35S.O.SCBV.PVY.
  • Figure 58 is a diagrammatic representation of the plasmid pART27.35S.O.SCBV.YVP.
  • Figure 59 is a diagrammatic representation of the plasmid pART7.PVYx2.
  • Figure 60 is a diagrammatic representation of the plasmid pART7.PVYx3.
  • Figure 61 is a diagrammatic representation of the plasmid pART7.PVYx4.
  • Figure 62 is a diagrammatic representation of the plasmid pART7.PVY.LNYV.PVY.
  • Figure 63 is a diagrammatic representation of the plasmid pART7.PVY.LNYV. YVP ⁇ .
  • Figure 64 is a diagrammatic representation of the plasmid pART7.PVY.LNYV.YVP.
  • Figure 65 is a diagrammatic representation of pART7.35S.PVY.SCBV.YVP.
  • Figure 66 is a diagrammatic representation of pART7.35S.PVYx3.SCBV.YVPx3.
  • Figure 67 is a diagrammatic representation of pART7.PVYx3.LNYV.YVPx3.
  • Figure 68 is a diagrammatic representation of the plasmid pART7.PVYMULTI. 15
  • the present invention provides a method of modulating the expression of a target gene in a cell, tissue or organ, said method at least comprising the step of introducing to said cell, tissue or organ one or more dispersed nucleic acid molecules or foreign nucleic acid molecules comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or complementary thereto for a time and under conditions sufficient for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
  • multiple copies is meant that two or more copies of the target gene are presented in close physical connection or juxtaposed, in the same or different orientation, on the same nucleic acid molecule, optionally separated by a stuffer fragment or intergenic region to facilitate secondary structure formation between each repeat where this is required.
  • the stuffer fragment may comprise any combination of nucleotide or amino acid residues, carbohydrate molecules or oligosaccharide molecules or carbon atoms or a homologue, analogue or derivative thereof which is capable of being linked covalently to a nucleic acid molecule.
  • the stuffer fragment comprises a sequence of nucleotides or a homologue, analogue or derivative thereof.
  • the stuffer fragment comprises a sequence of nucleotides of at least about 10-50 nucleotides in length, even more preferably at least about 50-100 nucleotides in length and still more preferably at least about 100-500 nucleotides in length.
  • dispersed or foreign nucleic acid molecule comprises intron/exon splice 16 -
  • the stuffer fragment may serve as an intron sequence placed between the 3'-splice site of the structural gene nearer the 5'-end of the gene and the 5'- splice site of the next downstream unit thereof.
  • the stuffer fragment placed there between should not include an in-frame translation stop codon, absent intron/exon splice junction sequences at both ends of the stuffer fragment or the addition of a translation start codon at the 5' end of each unit, as will be obvious to those skilled in the art.
  • Preferred stuffer fragments are those which encode detectable marker proteins or biologically-active analogues and derivatives thereof, for example luciferase, ⁇ - galacturonase, ⁇ -galactosidase, chloramphenicol acetyltransferase or green fluorescent protein, amongst others. Additional stuffer fragments are not excluded.
  • the detectable marker or an analogue or derivative thereof serves to indicate the expression of the synthetic gene of the invention in a cell, tissue or organ by virtue of its ability to confer a specific detectable phenotype thereon, preferably a visually-detectable phenotype.
  • modulating shall be taken to mean that expression of the target gene is reduced in amplitude and/or the timing of gene expression is delayed and/or the developmental or tissue-specific or cell-specific pattern of target gene expression is altered, compared to the expression of said gene in the absence of the inventive method described herein.
  • the present invention is directed to a modulation of gene expression which comprises the repression, delay or reduction in amplitude of target gene expression in a specified cell, tissue or organ of a eukaryotic organism, in particular a plant such as a monocotyledonous or dicotyledonous plant, or a human or other animal and even more particularly a 17
  • vertebrate and invertebrate animal such as an insect, aquatic animal (eg. fish, shellfish, mollusc, crustacean such as a crab, lobster or prawn, an avian animal or a mammal, amongst others).
  • an insect eg. fish, shellfish, mollusc, crustacean such as a crab, lobster or prawn, an avian animal or a mammal, amongst others.
  • target gene expression is completely inactivated by the dispersed nucleic acid molecules or foreign nucleic acid molecules which has been introduced to the cell, tissue or organ.
  • the reduced or eliminated expression of the target gene which results from the performance of the invention may be attributed to reduced or delayed translation of the mRNA transcription product of the target gene or alternatively, the prevention of translation of said mRNA, as a consequence of sequence-specific degradation of the mRNA transcript of the target gene by an endogenous host cell system.
  • sequence-specific degradation of the mRNA transcript of the target gene occurs either prior to the time or stage when the mRNA transcript of the target gene would normally be translated or alternatively, at the same time as the mRNA transcript of the target gene would normally be translated. Accordingly, the selection of an appropriate promoter sequence to regulate expression of the introduced dispersed nucleic acid molecule or foreign nucleic acid molecule is an important consideration to optimum performance of the invention. For this reason, strong constitutive promoters or inducible promoter systems are especially preferred for use in regulating expression of the introduced dispersed nucleic acid molecules or foreign nucleic acid molecules.
  • the present invention clearly encompasses reduced expression wherein reduced expression of the target gene is effected by lowered transcription, subject to the proviso that a reduction in transcription is not the sole mechanism by which this occurs and said reduction in transcription is at least accompanied by reduced translation of 18
  • the target gene may be a genetic sequence which is endogenous to the animal cell or alternatively, a non-endogenous genetic sequence, such as a genetic sequence which is derived from a virus or other foreign pathogenic organism and is capable of entering a cell and using the cell's machinery following infection.
  • the target gene is a non-endogenous genetic sequence to the animal cell, it is desirable that the target gene encodes a function which is essential for replication or reproduction of the viral or other pathogen.
  • the present invention is particularly useful in the prophylactic and therapeutic treatment of viral infection of an animal cell or for conferring or stimulating resistance against said pathogen.
  • the target gene comprises one or more nucleotide sequences of a viral pathogen of a plant or an animal cell, tissue or organ.
  • the viral pathogen may be a retrovirus, for example a lentivirus such as the immunodeficiency viruses, a single- stranded (+) RNA virus such as bovine enterovirus (BEV) or Sinbis alphavirus.
  • the target gene can comprise one or more nucleotide sequences of a viral pathogen of an animal cell, tissue or organ, such as but not limited to a double- stranded DNA virus such as bovine herpes virus or herpes simplex virus I (HSV I), amongst others.
  • the virus pathogen is preferably a potyvirus, caulimovirus, badnavirus, geminivirus, reovirus, rhabdovirus, bunyavirus, tospovirus, tenuivirus, tombusvirus, luteovirus, sobemovirus, bromovirus, cucomovirus, ilavirus, alfamovirus, tobamovirus, tobravirus, potexvirus and clostrovirus, such as but not limited to CaMV, SCSV, PVX, PVY, PLRV, and TMV, amongst others.
  • a potyvirus such as but not limited to CaMV, SCSV, PVX, PVY, PLRV, and TMV, amongst others.
  • virus- encoded functions may be complemented in trans by polypeptides encoded by the host cell.
  • the replication of the bovine herpes virus genome in the host cell may be facilitated by host cell DNA polymerases which are capable of complementing an inactivated viral DNA polymerase gene.
  • a further alternative embodiment of the invention provides for the target gene to encode a viral or foreign polypeptide which is not capable of being complemented by a host cell function, such as a virus-specific genetic sequence.
  • exemplary target genes according to this embodiment of the invention include, but are not limited to genes which encode virus coat proteins, uncoating proteins and RNA- dependent DNA polymerases and RNA-dependent RNA polymerases, amongst others.
  • the target gene is the BEV RNA-dependent RNA polymerase gene or a homologue, analogue or derivative thereof or PVY Nia protease-encoding sequences.
  • the cell in which expression of the target gene is modified may be any cell which is derived from a multicellular plant or animal, including cell and tissue cultures thereof.
  • the animal cell is derived from an insect, reptile, amphibian, bird, human or other mammal.
  • Exemplary animal cells include embryonic stem cells, cultured skin fibroblasts, neuronal cells, somatic cells, haematopoietic stem cells, T-cells and immortalised cell lines such as COS, VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, baby hamster kidney (BHK) or MDBK cell lines, amongst others.
  • Such cells and cell lines are readily available to those skilled in the art.
  • the tissue or organ in which expression of the target gene is modified may be any tissue or organ comprising such animal cells.
  • the plant cell is derived from a monocotyledonous or dicotyledonous plant 20
  • the term "dispersed nucleic acid molecule” shall be taken to refer to a nucleic acid molecule which comprises one or more multiple copies, preferably tandem direct repeats, of a nucleotide sequence which is substantially identical or complementary to the nucleotide sequence of a gene which originates from the cell, tissue or organ into which said nucleic acid molecule is introduced, wherein said nucleic acid molecule is non-endogenous in the sense that it is introduced to the cell, tissue or organ of an animal via recombinant means and will generally be present as extrachromosomal nucleic acid or alternatively, as integrated chromosomal nucleic acid which is genetically-unlinked to said gene.
  • the "dispersed nucleic acid molecule” will comprise chromosomal or extrachromosomal nucleic acid which is unlinked to the target gene against which it is directed in a physical map, by virtue of their not being tandemly-linked or alternatively, occupying a different chromosomal position on the same chromosome or being localised on a different chromosome or present in the cell as an episome, plasmid, cosmid or virus particle.
  • foreign nucleic acid molecule an isolated nucleic acid molecule which has one or more multiple copies, preferably tandem direct repeats, of a nucleotide sequence which originates from the genetic sequence of an organism which is different from the organism to which the foreign nucleic acid molecule is introduced.
  • This definition encompasses a nucleic acid molecule which originates from a different individual of the same lowest taxonomic grouping (i.e.
  • nucleic acid molecule which originates from a different individual of a different taxonomic grouping as the taxonomic grouping to which said nucleic acid molecule is introduced, such as a gene derived from a viral pathogen.
  • a target gene against which a foreign nucleic acid molecule acts in the performance of the invention may be a nucleic acid molecule which has been 21
  • Exemplary target genes according to this embodiment of the invention include the green fluorescent protein-encoding gene derived from the jellyfish Aequoria victoria (Prasher et a/., 1992; International Patent Publication No. WO 95/07463), tyrosinase genes and in particular the murine tyrosinase gene (Kwon et al., 1988), the Escherichia coli lac ⁇ gene which is capable of encoding a polypeptide repressor of the lacZ gene, the porcine ⁇ -1 ,3-galactosyltransferase gene (NCBI Accession No. L36535) exemplified herein, and the PVY and BEV structural genes exemplified herein or a homologue, analogue or derivative of said genes or a complementary nucleotide sequence thereto.
  • green fluorescent protein-encoding gene derived from the jellyfish Aequoria victoria (Prasher et a/., 1992;
  • the present invention is further useful for simultaneously targeting the expression of several target genes which are co-expressed in a particular cell, for example by using a dispersed nucleic acid molecule or foreign nucleic acid molecule which comprises nucleotide sequences which are substantially identical to each of said co-expressed target genes.
  • substantially identical is meant that the introduced dispersed or foreign nucleic acid molecule of the invention and the target gene sequence are sufficiently identical at the nucleotide sequence level to permit base-pairing there between under standard intracellular conditions.
  • the nucleotide sequence of each repeat in the dispersed or foreign nucleic acid molecule of the invention and the nucleotide sequence of a part of the target gene sequence are at least about 80-85% identical at the nucleotide sequence level, more preferably at least about 85-90% identical, even more preferably at least about 90-95% identical and still even more preferably at least about 95-99% or 100% identical at the nucleotide sequence level.
  • the present invention requires at least two copies of the target gene sequence to be expressed in the cell.
  • the multiple copies of the target gene sequence are presented in the dispersed nucleic acid molecule or the foreign nucleic acid molecule as tandem inverted repeat sequences and/or tandem direct repeat sequences.
  • Such configurations are exemplified by the "test plasmids" described herein that comprise Gait, BEV or PVY gene regions.
  • the dispersed or foreign nucleic acid molecule which is introduced to the cell, tissue or organ comprises RNA or DNA.
  • the dispersed or foreign nucleic acid molecule further comprises a nucleotide sequence or is complementary to a nucleotide sequence which is capable of encoding an amino acid sequence encoded by the target gene.
  • the nucleic acid molecule includes one or more ATG or AUG translational start codons.
  • Standard methods may be used to introduce the dispersed nucleic acid molecule or foreign nucleic acid molecule into the cell, tissue or organ for the purposes of modulating the expression of the target gene.
  • the nucleic acid molecule may be introduced as naked DNA or RNA, optionally encapsulated in a liposome, in a virus particle as attenuated virus or associated with a virus coat or a transport protein or inert carrier such as gold or as a recombinant viral vector or bacterial vector or as a genetic construct, amongst others.
  • Administration means include injection and oral ingestion (e.g. in medicated food material), amongst others. - 23
  • the subject nucleic acid molecules may also be delivered by a live delivery system such as using a bacterial expression system optimised for their expression in bacteria which can be incorporated into gut flora.
  • a viral expression system can be employed.
  • one form of viral expression is the administration of a live vector generally by spray, feed or water where an infecting effective amount of the live vector (e.g. virus or bacterium) is provided to the animal.
  • Another form of viral expression system is a non-replicating virus vector which is capable of infecting a cell but not replicating therein.
  • the non-replicating viral vector provides a means of introducing to the human or animal subject genetic material for transient expression therein.
  • the mode of administering such a vector is the same as a live viral vector.
  • the carriers, excipients and/or diluents utilised in delivering the subject nucleic acid molecules to a host cell should be acceptable for human or veterinary applications.
  • Such carriers, excipients and/or diluents are well-known to those skilled in the art.
  • Carriers and/or diluents suitable for veterinary use include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the composition is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the invention provides a method of modulating the expression of a target gene in a cell, tissue or organ, said method at least comprising the steps of: (i) selecting one or more dispersed nucleic acid molecules or foreign nucleic acid molecules which comprise multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or which is complementary thereto; and
  • nucleotide sequences for targeting expression of the target gene may be employed.
  • multiple copies of specific regions of characterised genes may be cloned in operable connection with a suitable promoter and assayed for efficacy in reducing target gene expression.
  • shotgun libraries comprising multiple copies of genetic sequences may be produced and assayed for their efficacy in reducing target gene expression.
  • the advantage associated with the latter approach is that it is not necessary to have any prior knowledge of the significance of any particular target gene in specifying an undesirable phenotype in the cell.
  • shotgun libraries comprising virus sub-genomic fragments may be employed and tested directly for their ability to confer virus immunity on the animal host cell, without prior knowledge of the role which any virus genes play in pathogenesis of the host cell.
  • shotgun library is a set of diverse nucleotide sequences wherein each member of said set is preferably contained within a suitable plasmid, cosmid, bacteriophage or virus vector molecule which is suitable for maintenance and/or replication in a cellular host.
  • shotgun library includes a representative library, in which the extent of diversity between the nucleotide sequences is numerous such that all sequences in the genome of the organism from which said nucleotide sequences is derived are present in the "set” or alternatively, a limited library in which there is a lesser degree of diversity between said sequences.
  • shotgun library further encompasses random nucleotide sequences, wherein the nucleotide sequence comprises viral or cellular genome fragments, amongst others obtained for example by shearing or partial digestion of genomic DNA using restriction endonucleases, amongst other approaches.
  • a "shotgun library” further includes cells, virus particles and bacteriophage particles comprising the - 25 -
  • Preferred shotgun libraries according to this embodiment of the invention are "representative libraries", comprising a set of tandem repeated nucleotide sequences derived from a viral pathogen of a plant or an animal.
  • the shotgun library comprises cells, virus particles or bacteriophage particles comprising a diverse set of tandem- repeated nucleotide sequences which encode a diverse set of amino acid sequences, wherein the member of said diverse set of nucleotide sequences are placed operably under the control of a promoter sequence which is capable of directing the expression of said tandem-repeated nucleotide sequence in the cell.
  • nucleotide sequence of each unit in the tandem-repeated sequence may comprise at least about 1 to 200 nucleotides in length.
  • the introduced nucleic acid molecule is preferably in an expressible form.
  • expressible form is meant that the subject nucleic acid molecule is presented in an arrangement such that it may be expressed in the cell, tissue, organ or whole organism, at least at the transcriptional level (i.e. it is expressed in the animal cell to yield at least an mRNA product which is optionally translatable or translated to produce a recombinant peptide, oligopeptide or polypeptide molecule).
  • a synthetic gene or a genetic construct comprising said synthetic gene is produced, wherein said synthetic gene comprises a nucleotide sequence as described supra in 26
  • the subject nucleic acid molecule will be operably connected to one or more regulatory elements sufficient for eukaryotic transcription to occur.
  • a further alternative embodiment of the invention provides a method of modulating the expression of a target gene in an animal cell, tissue or organ, said method at least comprising the steps of:
  • a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); and/or
  • a structural region corresponding to the coding regions optionally further comprising untranslated sequences and/or a heterologous promoter sequence which consists of transcriptional and/or translational regulatory regions capable of 27
  • gene is also used to describe synthetic or fusion molecules encoding all or part of a functional product, in particular a sense or antisense mRNA product or a peptide, oligopeptide or polypeptide or a biologically-active protein.
  • synthetic gene refers to a non-naturally occurring gene as hereinbefore defined which preferably comprises at least one or more transcriptional and/or translational regulatory sequences operably linked to a structural gene sequence.
  • structural gene shall be taken to refer to a nucleotide sequence which is capable of being transmitted to produce mRNA and optionally, encodes a peptide, oligopeptide, polypeptide or biologically active protein molecule.
  • Those skilled in the art will be aware that not all mRNA is capable of being translated into a peptide, oligopeptide, polypeptide or protein, for example if the mRNA lacks a functional translation start signal or alternatively, if the mRNA is antisense mRNA.
  • the present invention clearly encompasses synthetic genes comprising nucleotide sequences which are not capable of encoding peptides, oligopeptides, polypeptides or biologically-active proteins. In particular, the present inventors have found that such synthetic genes may be advantageous in modifying target gene expression in cells, tissues or organs of a prokaryotic or eukaryotic organism.
  • structural gene region refers to that part of a synthetic gene which comprises a dispersed nucleic acid molecule or foreign nucleic acid molecule as described herein which is expressed in a cell, tissue or organ under the control of a promoter sequence to which it is operably connected.
  • a structural gene region may comprise one or more dispersed nucleic acid molecules and/or foreign nucleic acid molecules operably under the control of a single promoter sequence or multiple promoter sequences.
  • the structural gene region of a synthetic gene may comprise a nucleotide sequence which is capable of encoding an amino acid 28 -
  • a structural gene region which is used in the performance of the instant invention may also comprise a nucleotide sequence which encodes an animo acid sequence yet lacks a functional translation initiation codon and/or a functional translation stop codon and, as a consequence, does not comprise a complete open reading frame.
  • the term "structural gene region" also extends to a non-coding nucleotide sequences, such as 5'- upstream or 3'-downstream sequences of a gene which would not normally be translated in a eukaryotic cell which expresses said gene.
  • a structural gene region may also comprise a fusion between two or more open reading frames of the same or different genes.
  • the invention may be used to modulate the expression of one gene, by targeting different non-contiguous regions thereof or alternatively, to simultaneously modulate the expression of several different genes, including different genes of a multigene family.
  • the fusion may provide the added advantage of conferring simultaneous immunity or protection against several pathogens, by targeting the expression of genes in said several pathogens.
  • the fusion may provide more effective immunity against any pathogen by targeting the expression of more than one gene of that pathogen.
  • Particularly preferred structural gene regions according to this aspect of the invention are those which include at least one translatable open reading frame, more preferably further including a translational start codon located at the 5'-end of said open reading frame, albeit not necessarily at the 5'-terminus of said structural gene region.
  • the structural gene region may comprise at least one translatable open reading frame and/or AUG or ATG translational start codon, the inclusion of such sequences in no way suggests that the present invention requires translation of the introduced nucleic acid molecule to occur in order to modulate the - 29 -
  • the inclusion of at least one translatable open reading frame and/or translational start codon in the subject nucleic acid molecule may serve to increase stability of the mRNA transcription product thereof, thereby improving the efficiency of the invention.
  • the optimum number of structural gene sequences to be included in the synthetic genes of the present invention may be determined empirically by those skilled in the art, without any undue experimentation and by following standard procedures such as the construction of the synthetic gene of the invention using recombinase-deficient cell lines, reducing the number of repeated sequences to a level which eliminates or minimises recombination events and by keeping the total length of the multiple structural gene sequence to an acceptable limit, preferably no more than 5-1 Okb, more preferably no more than 2-5kb and even more preferably no more than 0.5-2. Okb in length.
  • the structural gene region comprises more than one dispersed nucleic acid molecule or foreign nucleic acid molecule it shall be referred to herein as a "multiple structural gene region" or similar term.
  • the present invention clearly extends to the use of multiple structural gene regions which preferably comprise a direct repeat sequence, inverted repeat sequence or interrupted palindrome sequence of a particular structural gene, dispersed nucleic acid molecule or foreign nucleic acid molecule, or a fragment thereof.
  • Each dispersed or foreign nucleic acid molecule contained within the multiple structural gene unit of the subject synthetic gene may comprise a nucleotide sequence which is substantially identical to a different target gene in the same organism.
  • Such an arrangement may be of particular utility when the synthetic gene is intended to provide protection against a pathogen in a cell, tissue or organ, in particular a viral pathogen, by modifying the expression of viral target genes.
  • the multiple structural gene may comprise nucleotide sequences (i.e. two or more dispersed or foreign nucleic acid molecules) which are substantially identical to two or more target genes selected from the list comprising DNA polymerase, RNA polymerase, Nia protease, and coat protein or other target gene which is essential for viral infectivity, replication or reproduction.
  • the structural gene units are selected such that the target genes to which they are substantially identical are normally expressed at approximately the same time (or later) in an infected cell, tissue or organ as (than) the multiple structural gene of the subject synthetic gene is expressed under control of the promoter sequence.
  • the promoter controlling expression of the multiple structural gene will usually be selected to confer expression in the cell, tissue or organ over the entire life cycle of the virus when the viral target genes are expressed at different stages of infection.
  • the individual units of the multiple structural gene may be spatially connected in any orientation relative to each other, for example head-to-head, head-to-tail or tail-to-tail and all such configurations are within the scope of the invention.
  • the synthetic gene For expression in eukaryotic cells, the synthetic gene generally comprises, in addition to the nucleic acid molecule of the invention, a promoter and optionally other regulatory sequences designed to facilitate expression of the dispersed nucleic acid molecule or foreign nucleic acid molecule.
  • transcriptional regulatory sequences of a classical genomic gene including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • a promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene region, the expression of which it regulates.
  • the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell.
  • Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the sense molecule and/or to alter the spatial expression and/or temporal expression of said sense molecule.
  • regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense molecule, thereby conferring copper inducibility on the expression of said molecule.
  • Placing a dispersed or foreign nucleic acid molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous - 32
  • gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • promoters suitable for use in the synthetic genes of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in plant, animal, insect, fungal, yeast or bacterial cells.
  • the promoter may regulate the expression of the structural gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or pathogens, or metal ions, amongst others.
  • the promoter is capable of regulating expression of a nucleic acid molecule in a eukaryotic cell, tissue or organ, at least during the period of time over which the target gene is expressed therein and more preferably also immediately preceding the commencement of detectable expression of the target gene in said cell, tissue or organ.
  • promoters are particularly preferred for the purposes of the present invention or promoters which may be induced by virus infection or the commencement of target gene expression.
  • Plant-operable and animal-operable promoters are particularly preferred for use in the synthetic genes of the present invention.
  • preferred promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator- promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, CaMV 35S promoter, SCSV promoter, SCBV promoter and the like.
  • the promoter sequence is a constitutive strong promoter such as the CMV-IE promoter or the SV40 early promoter sequence, the SV40 late promoter sequence, the CaMV 35S promoter, or the SCBV promoter, amongst others.
  • a constitutive strong promoter such as the CMV-IE promoter or the SV40 early promoter sequence, the SV40 late promoter sequence, the CaMV 35S promoter, or the SCBV promoter, amongst others.
  • the terms "in operable connection with” or “operably under the control” or similar shall be taken to indicate that expression of the structural gene region or multiple structural gene region is under the control of the promoter sequence with which it is spatially connected; in a cell, tissue, organ or whole organism.
  • a structural gene region i.e. dispersed nucleic acid molecule or foreign nucleic acid molecule
  • multiple structural gene region is placed operably in connection with a promoter orientation relative to the promoter sequence, such that when it is transcribed an mRNA product is synthesized which, if translated, is capable of encoding a polypeptide product of the target gene or a fragment thereof.
  • the present invention is not to be limited to the use of such an arrangement and the invention clearly extends to the use of synthetic genes and genetic constructs wherein the a structural gene region or multiple structural gene region is placed in the "antisense" orientation relative to the promoter sequence, such that at least a part of the mRNA transcription product thereof is complementary to the mRNA encoded by the target gene or a fragment thereof.
  • dispersed nucleic acid molecule foreign nucleic acid molecule or multiple structural gene region comprises tandem direct and/or inverted repeat sequences of the target gene, all combinations of the above-mentioned configurations are encompassed by the invention.
  • the structural gene region or multiple structural gene region is operably connected to both a first promoter sequence and a second promoter sequence, wherein said promoters are located at the distal and proximal ends thereof such that at least one unit of said a structural gene region or multiple structural gene region is placed in the "sense" orientation relative to the first promoter sequence and in the "antisense” orientation relative to the second promoter sequence.
  • the first and second promoters be different, to prevent competition there between for cellular transcription factors which bind thereto. The advantage of this arrangement is that the effects of transcription from the first and second promoters in reducing target gene expression in the cell may be compared to determine the optimum orientation for each nucleotide sequence tested.
  • the synthetic gene preferably contains additional regulatory elements for efficient transcription, for example a transcription termination sequence.
  • Terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants or synthesized de novo.
  • the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used.
  • terminators particularly suitable for use in the synthetic genes of the present invention include the SV40 polyadenylation signal, the HSV TK polyadenylation signal, the CYC1 terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) gene terminator of Agrobactehum tumefaciens, the - 35 -
  • Cauliflower mosaic virus (CaMV) 35S gene the Cauliflower mosaic virus (CaMV) 35S gene
  • the zein gene terminator from Zea mays the Rubisco small subunit gene (SSU) gene terminator sequences
  • SCSV subclover stunt virus
  • any t ?o-independent E.coli terminator or the lacZ alpha terminator, amongst others.
  • the terminator is the SV40 polyadenylation signal or the HSVTK polyadenylation signal which are operable in animal cells, tissues and organs, octopine synthase (OCS) or nopaline synthase (NOS) terminator active in plant cells, tissues or organs, or the lacZ alpha terminator which is active in prokaryotic cells.
  • OCS octopine synthase
  • NOS nopaline synthase
  • a genetic construct which comprises two or more structural gene regions or multiple structural gene regions wherein each of said structural gene regions is placed operably under the control of its own promoter sequence.
  • the orientation of each structural gene region may be varied to maximise its modulatory effect on target gene expression.
  • the promoters controlling expression of the structural gene unit are preferably different promoter sequences, to reduce competition there between for cellular transcription factors and RNA polymerases.
  • Preferred promoters are selected from those referred to supra. - 36 -
  • the synthetic genes described supra are capable of being modified further, for example by the inclusion of marker nucleotide sequences encoding a detectable marker enzyme or a functional analogue or derivative thereof, to facilitate detection of the synthetic gene in a cell, tissue or organ in which it is expressed.
  • the marker nucleotide sequences will be present in a translatable format and expressed, for example as a fusion polypeptide with the translation product(s) of any one or more of the structural genes or alternatively as a non-fusion polypeptide.
  • the synthetic genes of the present invention may be introduced to a suitable cell, tissue or organ without modification as linear DNA in the form of a genetic construct, optionally contained within a suitable carrier, such as a cell, virus particle or liposome, amongst others.
  • a suitable carrier such as a cell, virus particle or liposome, amongst others.
  • the synthetic gene of the invention is inserted into a suitable vector or episome molecule, such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced.
  • a further aspect of the invention provides a genetic construct which at 37 -
  • Genetic constructs are particularly suitable for the transformation of a eukaryotic cell to introduce novel genetic traits thereto, in addition to the provision of resistance characteristics to viral pathogens.
  • additional novel traits may be introduced in a separate genetic construct or, alternatively on the same genetic construct which comprises the synthetic genes described herein.
  • an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to a eukaryotic cell, or integrated into the genome of a eukaryotic cell.
  • the origin of replication is functional in a bacterial cell and comprises the pUC or the ColE1 origin or alternatively the origin of replication is operable in a eukaryotic cell, tissue and more preferably comprises the 2 micron (2 ⁇ xn) origin of replication or the SV40 origin of replication.
  • selectable marker gene includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.
  • Suitable selectable marker genes contemplated herein include the ampiciliin-resistance 38
  • tetracycline-resistance gene Tc r
  • bacterial kanamycin-resistance gene Kan r
  • Zeocin is a drug of bleomycin family which is trademark of InVitrogen Corporation
  • AURI-C zeocin resistance gene
  • nptW neomycin phosphotransferase gene
  • hygromycin-resistance gene ⁇ -glucuronidase (GUS) gene
  • chloramphenicol acetyltransferase (CAT) gene green fluorescent protein- encoding gene or the luciferase gene, amongst others.
  • the selectable marker gene is the npfll gene or Kan r gene or green fluorescent protein (GFP)-encoding gene.
  • selectable marker genes useful in the performance of the present invention and the subject invention is not limited by the nature of the selectable marker gene.
  • the present invention extends to all genetic constructs essentially as described herein, which include further genetic sequences intended for the maintenance and/or replication of said genetic construct in prokaryotes or eukaryotes and/or the integration of said genetic construct or a part thereof into the genome of a eukaryotic cell or organism.
  • Additional means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCI 2 and variations thereof, in particular - 39 -
  • a microparticle is propelled into a cell to produce a transformed cell.
  • Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp etal. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050).
  • the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
  • microparticles suitable for use in such systems include 1 to 5 ⁇ m gold spheres.
  • the DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
  • the synthetic genes and genetic constructs described herein are adapted for integration into the genome of a cell in which it is expressed.
  • Those skilled in the art will be aware that, in order to achieve integration of a genetic sequence or genetic construct into the genome of a host cell, certain additional genetic sequences may be required. In the case of plants, left and right border sequences from the T-DNA of the Agrobactehum tumefaciens Ti plasmid will generally be required.
  • the present invention further extends to an isolated cell, tissue or organ comprising 40
  • the present invention extends further to regenerated tissues, organs and whole organisms derived from said cells, tissues and organs and to propagules and progeny thereof.
  • plants may be regenerated from transformed plant cells or tissues or organs on hormone-containing media and the regenerated plants may take a variety of forms, such as chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species).
  • Transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.
  • Plasmid pBluescript II (SK+) is commercially available from Stratagene and comprises the LacZ promoter sequence and /acZ-alpha transcription terminator, with a multiple cloning site for the insertion of structural gene sequences therein.
  • the plasmid further comprises the ColE1 and fl origins of replication and ampiciliin-resistance gene.
  • Plasmid pSVL is commercially-obtainable from Pharmacia and serves as a source of the SV40 late promoter sequence.
  • the nucleotide sequence of pSVL is also publicly available as GenBank Accession Number U13868.
  • Plasmid pCR2.1 is commercially available from Invitrogen and comprises the LacZ promoter sequence and lacZ- ⁇ transcription terminator, with a cloning site for the insertion of structural gene sequences there between. Plasmid pCR2.1 is designed to clone nucleic acid fragments by virtue of the A-overhang frequently synthesized by Taq polymerase during the polymerase chain reaction. PCR fragments cloned in this fashion are flanked by two EcoRI sites. The plasmid further comprises the ColE1 and fl origins of replication and kanamycin-resistance and ampiciliin-resistance genes.
  • Plasmid pEGFP-N1 MCS ( Figure 1; Clontech) contains the CMV IE promoter operably connected to an open reading frame encoding a red-shifted variant of wild-type green fluorescent protein (GFP; Prasher et al., 1992; Chalfie et al., 1994; Inouye and Tsuji, - 42 -
  • Plasmid pEGFP-N1 MCS contains a multiple cloning site comprising BglW and Bam ⁇ sites and many other restriction endonuclease cleavage sites, located between the CMV IE promoter and the GFP open reading frame. Structural genes cloned into the multiple cloning site will be expressed at the transcriptional level if they lack a functional translation start site, however such structural gene sequences will not be expressed at the protein level (i.e. translated).
  • Structural gene sequences inserted into the multiple cloning site which comprise a functional translation start site will be expressed as GFP fusion polypeptides if they are cloned in-frame with the GFP-encoding sequence.
  • the plasmid further comprises an SV40 polyadenylation signal downstream of the GFP open reading frame to direct proper processing of the 3'-end of mRNA transcribed from the CMV-IE promoter sequence.
  • the plasmid further comprises the SV40 origin of replication functional in animal cells; the neomycin-resistance gene comprising SV40 early promoter (SV40 EP in Figure 1) operably connected to the neomycin/kanamycin-resistance gene derived from Tn5 (Kan/neo in Figure 1) and the HSV thymidine kinase polyadenylation signal (HSV TK poly (A) in Figure 1), for selection of transformed cells on kamanycin, neomycin or G418; the pUC19 origin of replication which is functional in bacterial cells (pUC Ori in Figure 1); and the f1 origin of replication for single-stranded DNA production (fl Ori in Figure 1).
  • SV40 EP SV40 early promoter
  • HSV TK poly (A) HSV thymidine kinase polyadenylation signal
  • Plasmid pCMV.cass ( Figure 2) is an expression cassette for driving expression of a structural gene sequence under control of the CMV-IE promoter sequence.
  • Plasmid pCMV.cass was derived from pEGFP-N1 MCS by deletion of the GFP open reading frame as follows: Plasmid pEGFP-N1 MCS was digested with PinA and Not I, blunt- ended using Pfu ⁇ polymerase and then re-ligated. Structural gene sequences are cloned into pCMV.cass using the multiple cloning site, which is identical to the multiple cloning site of pEGFP-N1 MCS, except it lacks the P/ ' nAI site. 43
  • Plasmid pCMV.SV40L.cass ( Figure 3) comprises the synthetic poly A site and the SV40 late promoter sequence from plasmid pCR.SV40L ( Figure 4), sub-cloned as a Sal I fragment, into the Sal I site of plasmid pCMV.cass ( Figure 2), such that the CMV- IE promoter and SV40 late promoter sequences are capable of directing transcription in the same direction.
  • the synthetic poly(A) site at the 5' end of the SV40 promoter sequence is used as a transcription terminator for structural genes expressed from the CMV IE promoter in this plasmid, which also provides for the insertion of said structural gene via the multiple cloning site present between the SV40 late promoter and the synthetic poly(A) site ( Figure 5).
  • the multiple cloning sites are located behind the CMV-IE and SV40 late promoters, including Bam ⁇ and BglW sites.
  • Plasmid pCMV.SV40LR.cass ( Figure 4) comprises the SV40 late promoter sequence derived from plasmid pCR.SV40L, sub-cloned as a Sail fragment into the Sa/I site of the plasmid pCMV.cass ( Figure 2), such that the CMV-IE or the SV40 late promoter may drive transcription of a structural gene or a multiple structural gene unit, in the sense or antisense orientation, as desired.
  • a multiple cloning site is positioned between the opposing CMV- IE and SV40 late promoter sequences in this plasmid to facilitate the introduction of a structural gene sequence.
  • the structural gene sequence or multiple structural gene unit which is to be introduced into pCMV.SV40LR.cass will comprise both a 5' and a 3' polyadenylation signal as follows:
  • the structural gene sequence or multiple structural gene unit is to be expressed in the sense orientation from the CMV IE promoter sequence and/or in the antisense orientation from the SV40 late promoter, the 5' polyadenylation signal will be in the antisense orientation and the 3' 44
  • polyadenylation signal will be in the sense orientation; and (ii) where the structural gene sequence or multiple structural gene unit is to be expressed in the antisense orientation from the CMV IE promoter sequence and/or in the sense orientation from the SV40 late promoter, the 5' polyadenylation signal will be in the sense orientation and the 3' polyadenylation signal will be in the antisense orientation.
  • suitably-oriented terminator sequences may be placed at the 5'-end of the CMV and SV40L promoters, as shown in Figure 4.
  • plasmid pCMV.SV40LR.cass is further modified to produce a derivative plasmid which comprises two polyadenylation signals located between the CMV IE and SV40 late promoter sequences, in approriate orientations to facilitate expression of any structural gene located therebetween in the sense or antisense orientation from either the CMV IE promoter or the SV40 promoter sequence.
  • the present invention clearly encompasses such derivatives.
  • appropriately oriented terminators could be placed upstream of the CMV and SV40L promoters such that transcriptional termination could occur after readthrough of each of the two promoters in the antisense orientation.
  • Plasmid pCR.Bgl-GFP-Bam ( Figure 5) comprises an internal region of the GFP open reading frame derived from plasmid pEGFP-N1 MCS ( Figure 1) placed operably under the control of the lacZ promoter. To produce this plasmid, a region of the GFP open reading frame was amplified from pEGFP-N1 MCS using the amplification primers Bgl- GFP and GFP-Bam and cloned into plasmid pCR2.1. The internal GFP-encoding region in plasmid pCR.Bgl-GFP-Bam lacks functional translational start and stop codons. - 45 -
  • EGFP ( Figure 6) comprises the EGFP open reading frame derived from plasmid pEGFP-N1 MCS ( Figure 1) placed operably under the control of the lacZ promoter. To produce this plasmid, the EGFP encoding region of pEGFP-N1 MCS was excised as a NotMXho ⁇ fragment and cloned into the NotMXho cloning sites of plasmid pBluescript II (SK+).
  • Plasmid pCMV.EGFP ( Figure 7) is capable of expressing the EGFP structural gene under the control of the CMV-IE promoter sequence.
  • the EGFP sequence from plasmid pBSII(SK+).EGFP was excised as BamHUSacl fragment and cloned into the BglU/Sacl sites of plasmid pCMV.cass ( Figure 2).
  • Plasmid pCR.SV40L ( Figure 8) comprises the SV40 late promoter derived from plasmid pSVL (GenBank Accession No. U 13868; Pharmacia), cloned into pCR2.1 (Stratagene). To produce this plasmid, the SV40 late promoter was amplified using the primers SV40-1 and SV40-2 which comprise Sal I cloning sites to facilitate sub- cloning of the amplified DNA fragment into pCMV.cass. The primer also contains a synthetic poly (A) site at the 5' end, such that the amplicification product comprises the synthetic poly(A) site at the 5' end of the SV40 promoter sequence.
  • the BEV RNA-dependent RNA polymerase coding region was amplified as a 1 ,385 bp DNA fragment from a full-length cDNA clone encoding same, using primers designated BEV-1 and BEV-2, under standard amplification conditions.
  • the amplified DNA contained a 5'-Bgl II restriction enzyme site, derived from the BEV-1 primer sequence and a 3' ⁇ amHI restriction enzyme site, derived from the BEV-2 primer sequence.
  • the BEV-1 primer sequence contains a translation start signal 5'-ATG-3' engineered at positions 15-17, the amplified BEV polymerase 46 -
  • the amplified BEV polymerase structural gene comprises the start site in-frame with BEV polymerase-encoding nucleotide sequences,
  • the amplified BEV polymerase structural gene comprises the ATG start codon immediately upstream (ie. juxtaposed) to the BEV polymerase- encoding sequence.
  • This plasmid is present as Figure 9.
  • the complete BEV polymerase coding region was amplified from a full-length cDNA clone encoding same, using primers BEV-1 and BEV-3.
  • Primer BEV-3 comprises a BamHl restriction enzyme site at positions 5 to 10 inclusive and the complement of a translation stop signal at positions 11 to 13.
  • an open reading frame comprising a translation start signal and translation stop signal, contained between the Bgl II and BamHl restriction sites.
  • the amplified fragment was cloned into pCR2J (Stratagene) to produce plasmid pCR2.BEV.2 ( Figure 10).
  • a non-translatable BEV polymerase structural gene was amplified from a full-length BEV polymerase cDNA clone using the amplification primers BEV-3 and BEV-4.
  • Primer BEV-4 comprises a Bglll cloning site at positions 5-10 and sequences downstream of this Bglll site are homologous to nucleotide sequences of the BEV polymerase gene.
  • the BEV polymerase is expressed as part of a polyprotein and, as a consequence, there is no ATG translation start site in this gene.
  • the amplified DNA was cloned into plasmid pCR2.1 (Stratagene) to yield plasmid pCR.BEV.3 ( Figure 11).
  • Plasmid pCMV.EGFP.BEV2 ( Figure 12) was produced by cloning the BEV polymerase sequence from pCR.BEV.2 as a Bglll/BamHI fragment into the BamHl site of pCMV.EGFP. 47
  • Plasmid pCMV.BEV.2 ( Figure 13) is capable of expressing the entire BEV polymerase open reading frame under the control of CMV-IE promoter sequence.
  • the BEV polymerase sequence from pCR.BEV.2 was sub-cloned in the sense orientation as a Bg/ll-to-SamHI fragment into Sg/ll/SamHI-digested pCMV.cass ( Figure 2).
  • Plasmid pCMV.BEV.3 ( Figure 14) expresses a non-translatable BEV polymerase structural gene in the sense orientation under the control of the CMV-IE promoter sequence.
  • the BEV polymerase sequence from pCR.BEV.3 was sub-cloned in the sense orientation as a Sg/ll-to-SamHI fragment into Bg/ll/ ⁇ amHI-digested pCMV.cass ( Figure 2).
  • Plasmid pCMV.VEB ( Figure 15) expresses an antisense BEV polymerase mRNA under the control of the CMV-IE promoter sequence.
  • the BEV polymerase sequence from pCR.BEV.2 was sub-cloned in the antisense orientation as a Sg/ll-to-SamHI fragment into ⁇ g/ll/SamHI-digested pCMV.cass ( Figure 2).
  • Plasmid pCMV.BEV.GFP ( Figure 16) was constructed by cloning the GFP fragment from pCR.Bgl-GFP-Bam as a Bglll/BamHI fragment into BamHI-digested pCMV.BEV.2. This plasmid serves as a control in some experiments and also as an intermediate construct.
  • Plasmid pCMV.BEV.SV40-L ( Figure 17) comprises a translatable BEV polymerase 48 -
  • the BEV polymerase structural gene was sub-cloned as a Bg/ll-to-BamHI fragment into Bg/ll-digested pCMV.SV40L.cass DNA.
  • Plasmid pCMV.O.SV40L.BEV ( Figure 18) comprises a translatable BEV polymerase structural gene derived from plasmid pCR.BEV.2 cloned downstream of tandem CMV- IE promoter and SV40 late promoter sequences present in plasmid pCMV.SV40L.cass.
  • the BEV polymerase structural gene was sub-cloned in the sense orientation as a Bg/ll-to-BamHI fragment into BamHI-digested pCMV.SV40L.cass DNA.
  • Plasmid pCMV.O.SV40L.VEB ( Figure 19) comprises an antisense BEV polymerase structural gene derived from plasmid pCR.BEV.2 cloned downstream of tandem CMV- IE promoter and SV40 late promoter sequences present in plasmid pCMV.SV40L.cass.
  • the BEV polymerase structural gene was sub-cloned in the antisense orientation as a Bg/ll-to-BamHI fragment into BamHI-digested pCMV.SV40L.cass DNA.
  • Plasmid pCMV.BEVx2 ( Figure 20) comprises a direct repeat of a complete BEV polymerase open reading frame under the control of the CMV-IE promoter sequence. In eukaryotic cells at least, the open reading frame located nearer the CMV-IE promoter is translatable.
  • the BEV polymerase structural gene from plasmid pCR.BEV.2 was sub-cloned in the sense orientation as a Bg/ll-to- BamHl fragment into BamHI-digested pCMV.BEV.2, immediately downstream of the 49 -
  • Plasmid pCMV.BEVx3 ( Figure 21) comprises a direct repeat of three complete BEV polymerase open reading frames under the control of the CMV-1E promoter.
  • the BEV polymerase fragment from pCR.BEV.2 was cloned in the sense orientation as a Bglll/BamHI fragment into the BamHl site of pCMV.BEVx2, immediately downstream of the BEV polymerase sequences already present therein.
  • Plasmid pCMV.BEVx4 ( Figure 22) comprises a direct repeat of four complete BEV polymerase open reading frames under the control of the CMV-1 E promoter.
  • the BEV polymerase fragment from pCR.BEV.2 was cloned in the sense orientation as a Bglll/BamHI fragment into the BamHl site of pCMV.BEVx3, immediately downstream of the BEV polymerase sequences already present therein.
  • Plasmid pCMV.BEV.SV40L.BEV comprises a multiple structural gene unit comprising two BEV polymerase structural genes placed operably and separately under control of the CMV-IE promoter and SV40 late promoter sequences.
  • the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned in the sense orientation as a Bg/ll-to- BamHl fragment behind the SV40 late promoter sequence present in BamHI-digested pCMV.BEV.SV40L-O.
  • Plasmid pCMV.BEV.SV40L.VEB ( Figure 24) comprises a multiple structural gene unit comprising two BEV polymerase structural genes placed operably and separately - 50 -
  • the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned in the antisense orientation as a Bg/ll-to- BamHI fragment behind the SV40 late promoter sequence present in BamHI-digested pCMV.BEV.SV40L-O.
  • the BEV polymerase structural gene is expressed in the sense orientation under control of the CMV-IE promoter to produce a translatable mRNA, whilst the BEV polymerase structural gene is also expressed under control of the SV40 promoter to produce an antisense mRNA species.
  • Plasmid pCMV.BEV.GFP.VEB ( Figure 25) comprises a BEV structural gene inverted repeat or palindrome, interrupted by the insertion of a GFP open reading frame (stuffer fragment) between each BEV structural gene sequence in the inverted repeat.
  • the GFP stuffer fragment from pCR.Bgl-GFP- Bam was first sub-cloned in the sense orientation as a Bg/ll-to-BamHI fragment into BamHI-digested pCMV.BEV.2 to produce an intermediate plasmid pCMV.BEV.GFP wherein the BEV polymerase-encoding and GFP-encoding sequences are contained within the same 5'-Bg/ll-to-BamHI-3' fragment.
  • the BEV polymerase structural gene from pCMV.BEV.2 was then cloned in the antisense orientation as a Bg/ll-to-BamHI fragment into BamHI-digested pCMV.BEV.GFP.
  • the BEV polymerase structural gene nearer the CMV-IE promoter sequence in plasmid pCMV.BEV.GFP.VEB is capable of being translated, at least in eukaryotic cells.
  • Plasmid pCMV.EGFP.BEV2.PFG ( Figure 26) comprise a GFP palindrome, interrupted by the insertion of a BEV polymerase sequence between each GFP structural gene in the inverted repeat. To produce this plasmid the GFP fragment from pCR.Bgl-GFP- Bam was cloned as a Bglll/BamHI fragment into the BamHl site of pCMV.EGFP.BEV2 in the antisense orientation relative to the CMV promoter. - 51 -
  • Plasmid pCMV.BEV.SV40LR ( Figure 27) comprises a structural gene comprising the entire BEV polymerase open reading frame placed operably and separately under control of opposing CMV-IE promoter and SV40 late promoter sequences, thereby potentially producing BEV polymerase transcripts at least from both strands of the full- length BEV polymerase structural gene.
  • the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned, as a Bg/ll-to-BamHI fragment, into the unique Bglll site of plasmid pCMV.SV40LR.cass, such that the BEV open reading frame is present in the sense orientation relative to the CMV-IE promoter sequence.
  • EXAMPLE 2 Genetic constructs comprising the porcine ⁇ -1,3-galactosyltransferase (Gait) structural gene sequence or sequences operably connected to the CMV promoter sequence and/or the SV40L promoter sequence
  • Plasmid pcDNA3 is commercially available from Invitrogen and comprises the CMV-IE promoter and BGHpA transcription terminator, with multiple cloning sites for the insertion of structural gene sequences there between.
  • the plasmid further comprises the ColE1 and fl origins of replication and neomycin-resistance and ampiciliin- resistance genes.
  • Plasmid pcDNA3.Galt (BresaGen Limited, South Australia, Australia; Figure 28) is plasmid pcDNA3 (Invitrogen) and comprises the cDNA sequence encoding porcine gene alpha-1,3-galactosyltransferase (Gait) operably under the control of the CMV-IE promoter sequence such that it is capable of being expressed therefrom.
  • porcine gene alpha-1 ,3-galactosyltransferase cDNA was cloned as an EcoRI fragment into the EcoRI cloning site of pcDNA3.
  • the plasmid further comprises the ColE1 and fl origins of replication and the neomycin and ampiciliin-resistance genes.
  • Plasmid pCMV.Galt ( Figure 29) is capable of expressing the Gait structural gene under the control of the CMV-IE promoter sequence.
  • Gait sequence from plasmid pcDNA3.Galt was excised as an EcoRI fragment and cloned in the sense orientation into the EcoRI site of plasmid pCMV.cass ( Figure 2).
  • Plasmid pCMV.EGFP.Galt ( Figure 30) is capable of expressing the Gait structural gene as a Gait fusion polypeptide under the control of the CMV-IE promoter sequence.
  • the Gait sequence from pCMV.Galt ( Figure 29) was excised as a Bglll/BamHI fragment and cloned into the BamHl site of pCMV.EGFP.
  • Plasmid pCMV.Galt.GFP ( Figure 31) was produced by cloning the Gait cDNA as an EcORI fragment from pCDNA3 into EcoRI-digested pCMV.EGFP in the sense orientation. This plasmid serves as both a control and construct intermediate. 53
  • the plasmid pCMV.Galt.SV40L.0 ( Figure 32) comprises a Gait structural gene cloned downstream of the CMV promoter present in pCMV.SV40L.cass.
  • the Gait cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHI into Bglll-digested pCMV.SV40L.cass in the sense orientation.
  • the plasmid pCMV.O.SV40L.tlaG ( Figure 33) comprises a Gait structural gene clones in an antisense orientation downstream of the SV40L promoter present in pCMV.SV40L.cass.
  • the Gait cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHI into BamHI-digested pCMV.SV40L.cass in the antisense orientation.
  • Plasmid pCMV.O.SV40L.Galt ( Figure 34) comprises a Gait structural gene cloned downstream of the SV40L promoter present in pCMV.SV40L.cass. To produce the plasmid the Gait cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHI into BamHI-digested pCMV.SV40L.cass in the sense orientation.
  • Plasmid pCMV.Galtx2 ( Figure 35) comprises a direct repeat of a Gait open reading frame under the control of the CMV-IE promoter sequence. In eukaryotes cells at least, the open reading frame located nearer the CMV-IE promoter is translatable.
  • the Gait structural gene from pCMV.Galt was excised as a Bglll/BamHI fragment and cloned in the sense orientation into the BamHl cloning site of pCMV.Galt.
  • Plasmid pCMV.Galtx4 ( Figure 36) comprises a quadruple direct repeat of a Gait open - 54 -
  • the Galtx2 sequence from pCMV.Galtx2 was excised as a Bglll/BamHI fragment and cloned in the sense orientation into the BamHl cloning site of pCMV.Galtx2.
  • the plasmid pCMV.Galt.SV40L.Galt ( Figure 37) is designed to express two sense transcripts of Gait, one driven by the CMV promoter, the other by the SV40L promoter.
  • a Gait cDNA fragment from pCMV.Galt was cloned as a
  • the plasmid pCMV.Galt.SV40.tlaG ( Figure 38) is designed to express a sense transcript of Gait driven by the CMV promoter and an antisense transcript driven by the SV40L promoter.
  • a Gait cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHI fragment into Bglll-digested pCMV.O.SV40.talG in the sense orientation.
  • Plasmid pCMV.Galt.GFP.tlaG ( Figure 39) comprise a Gait palindrome, interrupted by the insertion of a GFP sequence between each Gait structural gene in the inverted repeat.
  • the Bglll/BamHI Gait cDNA fragment from pCMV.Galt was cloned into the BamHl site of pCMV.Galt.GFP in the antisense relative to the CMV promoter.
  • the plasmid pCMV.EGFP.Galt.PFG ( Figure 40) comprises a GFP palindrome, interrupted by the insertion of a Gait sequence between each GFP structural gene of the inverted repeat, expression of which is driven by the CMV promoter.
  • this plasmid the Gait sequences from pCMV.Galt were cloned as a Bglll/BamHI fragment into BamHI-digested pCMV.EGFP in the sense orientation to produce the intermediate pCMV.EGFP.Galt (not shown); following this further GFP sequences from pCR.Bgl-pCMV.EGFP.Galt in the antisense orientation.
  • the plasmid pCMV.Galt.SV40LR ( Figure 41) is designed to express GalT cDNA sequences cloned between the opposing CMV and SV40L promoters in the expression cassette pCMV.SV40LR.cass.
  • To produce this plasmid Gait sequences from pCMV.Galt were cloned as a Bglll/BamHI fragment in Bg Ill-digested pCMV.SV40LR.cass in the sense orientation relative to the 35S promoter.
  • EXAMPLE 3 Genetic constructs comprising PVY Nia sequences operably linked to the35S promoter sequence and/or the SCBV promoter sequence
  • Plasmid pART27 is a binary vector, specifically designed to be compatible with the pART7 expression cassette. It contains bacterial origins of replication for both E. coli and Agrobacterium tumefaciens, a spectinomycin resistance gene for bacterial selection, left and right T-DNA borders for transfer of DNA from Agrobacterium to plant cells and a kanamycin resistance cassette to permit selection of transformed plant cells.
  • the kanamycin resistance cassette is located between the T-DNA borders
  • pART27 also contains a unique NotI restriction site which permits cloning of constructs prepared in vectors such as pART7 to be cloned between the T-DNA borders. Construction of pART27 is described in Gleave, AP (1992).
  • 35S promoter in the described pART7 constructs was chosen; this was done to minimise any experimental artefacts that may arise from comparing different constructs with different insert orientations.
  • Plasmid pBC Plasmid pBC (KS-)
  • Plasmid pBC (KS-) is commercially available from Stratagene and comprises the LacZ promoter sequence and lacZ-alpha transcription terminator, with a multiple cloning site for the insertion of structural gene sequences therein.
  • the plasmid further comprises the ColE1 and fl origins of replication and a chloroamphenicol-resistance gene.
  • Plasmid pSP72 is commercially available from Promega and contains a multiple cloning site for the insertion of structural gene sequences therein.
  • the plasmid further comprises the ColE1 origin of replication and an ampiciliin-resistance gene.
  • Plasmid pART7 is an expression cassette designed to drive expression of sequences cloned behind the 35S promoter. It contains a polylinkerto assist cloning and a region of the octipine synthase terminator.
  • the 35S expression cassette is flanked by two Not I restriction sites which permits cloning into binary expression vectors, such as pART27 which contains a unique NotI site. Its construction as described in Gleave, AP (1992), a map is shown in Figure 43.
  • Plasmid p35S.CMV.cass was designed to express two separate gene sequences cloned into a single plasmid. To create this plasmid, sequences corresponding to the nos terminator and the SCBV promoter were amplified by PCR then cloned in the polylinker of pART7 between the 35S promoter and OCS. - 57 -
  • the resulting plasmid has the following arrangement of elements:
  • sequences cloned into polylinker 1 is controlled by the 35S promoter
  • expression of sequences cloned into polylinker 2 is controlled by the SCBV promoter.
  • NOS terminator sequences were amplified from the plasmid pAHC27 (Christensen and Quail, 1996) using the two oligonucleotides;
  • Nucleotide residues 1 to 17 for NOS 5' and 1 to 15 for NOS 3' represent additional nucleotides designed to assist in construct preparation by adding additional restriction sites.
  • Nucleotide residues 1 to 17 for NOS 5' and 1 to 15 for NOS 3' represent additional nucleotides designed to assist in construct preparation by adding additional restriction sites.
  • For NOS 5' these are BamHl, Smal, Aatll and the first 4 bases of an Nrul site, for NOS 3' these are Ncol and Sfil sites.
  • the remaining sequences for each oligonucleotide are homologous to the 5' and 3' ends respectively of NOS sequences in pAHC 27.
  • SCBV promoter sequences were amplified from the plasmid pScBV-20 (Tzafir et al, 1998) using the two oligonucleotides:
  • SCBV 5' 5'-CCATGGCCTATATGGCCATTCCCCACATTCAAG-3';
  • SCBV 3' 5'-AACGTTAACTTCTACCCAGTTCCAGAG-3' 58
  • Nucleotide residues 1 to 17 of SCBV 5' encode Ncol and Sfil restriction sites designed to assist in construct preparation, the remaining sequences are homologous to upstream sequences of the SCMV promoter region.
  • Nucleotide residues 1 to 9 of SCBV 3' encode Psp10461 and Hpal restriction sites designed to assist in construct preparation, the remaining sequences are homologous to the reverse and complement of sequences near the transcription initiation site of SCBV.
  • a region of the PVY genome was amplified by PCR using reverse-transcribed RNA isolated from PVY-infected tobacco as a template using standard protocols and cloned into a plasmid pGEM 3 (Stratagene), to create pGEM.PVY.
  • pGEM 3 (Stratagene)
  • a Sall/Hindlll fragment from pGEM.PVY, corresponding to a Sall/Hindlll fragment positions 1536-2270 of the PVY strain O sequence was then subcloned into the plasmid pBC (Stratagene Inc.) to create pBC.PVY ( Figure 44).
  • Plasmid pSP72.PVY was prepared by inserting an EcoRI/Sall fragment from pBC.PVY into EcoRI/Sall cut ⁇ SP72 (Promega). This construct contains additional restriction sites flanking the PVY insert which were used to assist subsequent manipulations. A map of this construct is shown in Figure 45.
  • Plasmid ClapBC.PVY Plasmid Cla pBC.PVY was prepared by inserting a Clal/Sall fragment from pSP72.PVY - 59 -
  • Plasmid pBC.PVYx2 contains two direct head-to-tail repeats of the PVY sequences derived from pBC.PVY.
  • the plasmid was generated by cloning an Accl/Clal PVY fragment from pSP72.PVY into Accl cut pBC.PVY and is shown in Figure 47.
  • Plasmid pSP72.PVYx2 contains two direct head-to-tail repeats of the PVY sequences derived from pBC.PVY.
  • the plasmid was generated by cloning an Accl/Clal PVY fragment from pBc.PVY into Accl cut pSP72.PVY and is shown in Figure 48.
  • Plasmid pBC.PVYx3 contains three direct head-to-tail repeats of the PVY sequences derived from pBC.PVY.
  • the plasmid was prepared by cloning an Accl/Clal PVY fragment from pSP72.PVY into Accl cut pBC.PVYx2 and is shown in Figure 49.
  • Plasmid pBC.PVYx4 contains four direct head-to-tail repeats of the PVY sequences derived from pBC.PVY.
  • the plasmid was prepared by cloning the direct repeat of PVY sequences from pSP72.PVYx2 as an Accl/Clal fragment into Accl cut pBC.PVYx2 and is shown in Figure 50.
  • the first 9 nucleotide of these primers encode a BamHl site, the remaining nucleotides are homologous to sequences of the LNYV 4b gene.
  • the plasmid pBC.PVY. LNYV. YVP contains an interrupted direct repeat of PVY sequences, to create this plasmid a Hpal/Hincll fragment from pSP72 was cloned into Smal-digested pBC.PVY.LNYV and a plasmid containing the sense orientation isolated, a map of this construct is shown in Figure 52.
  • the plasmid PBV.PVY.LNYV.YVP ⁇ contains a partial interrupted palindrome of PVY sequences.
  • One arm of the palindrome contains all the PVY sequences from pBC.PVY, the other arm contains part of the sequences from PVY, corresponding to sequences between the EcoRV and Hindi sites of pSP72.PVY.
  • Plasmid pBC.PVY.LNYV. YVP contains an interrupted palindrome of PVY sequences.
  • a Hpal/Hincll fragment from pSP72. was cloned into Sma-digested pBC.PVY.LNYV and a plasmid containing the antisense orientation isolated, a map of this construct is shown in Figure 54.
  • Plasmid pART7.PVY ( Figure 55) was designed to express PVY sequences driven by the 35S promoter. This plasmid serves as a control construct in these experiments, against which all other constructs was compared. To generate this plasmid a Clal/Accl fragment from ClapBC.PVY was cloned into Clal-digested pART7 and a plasmid with expected to express a sense PVY sequence with respect to the PVY genome, was selected.
  • Plasmid pART7.35S.PVY.SCBV.O ( Figure 56) was designed to act as a control for co- expression of multiple constructs from a single plasmid in transgenic plants.
  • the 35S promoter was designed to express PVY sense sequences, whilst the SCBV promoter was empty.
  • the PVY fragment from Cla pBC.PVY was cloned as a Xhol/EcoRI fragment into Xhol/EcoRI-digested pART7.35S.SCBV.cass to create p35S.PVY.SCBV>O.
  • Plasmid pART27.35S.O.SCBV.PVY ( Figure 57) was designed to act as an additional control for co-expression of multiple constructs from a single plasmid in transgenic plants. No expressible sequences were cloned behind the 35S promoter, whilst the SCBV promoter drove expression of a PVY sense fragment. To generate this plasmid, the PVY fragment from Cla pBC.PVY was cloned as a Clal fragment into Clal-digested pART7.35S.SCBV.cass, a plasmid containing PVY sequences in a sense orientation was isolated and designated p35S.O.SCBV.PVY.
  • Plasmids pART7.35S.O.SCBV.YVP & pART7.35S.O.SCBV.YVP Plasmid pART7.35S.O.SCBV.YVP ( Figure 58) was designed to act as an additional control for co-expression of multiple constructs from a single plasmid in transgenic plants. No expressible sequences were cloned behind the 35S promoter, whilst the SCBV promoter drove expression of a PVY antisense fragment.
  • the PVY fragment from Cla pBC.PVY was cloned as a Clal fragment into Clal- digested p35S.SCBV.cass, a plasmid containing PCY sequences in an antisense orientation was isolated and designated p35S.O.SCBV.YVP.
  • Sequences, consisting of the 35S promoter and NOS terminator, the SCBV promoter driving sense PVY sequences and the OCS terminator were excised as a NotI fragment and cloned into pART27, a plasmid with the desired insert orientation was isolated and designated pART27.35S.O.SCBV.YVP.
  • Plasmid pART7.PVYx2 ( Figure 59) was designed to express a direct repeat of PVY sequences driven by the 35S promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx2 were cloned as a Xhol/BamHI fragment into Xhol/BamHI cut pART7. Sequences consisting of the 35 S promoter, direct repeats of PVY and the OCS terminator were excised as a NotI fragment from pART7.PVYx2 and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.PVYx2.
  • Plasmid pART7.PVYx3 & pART27.PVYx3 Plasmid pART7.PVYx3 ( Figure 60) was designed to express a direct repeat of three PVY sequences driven by the 35S promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx3 were cloned as a Xhol/BamHI fragment into Xhol/BamHI cut pART7.
  • Plasmid pART7.PVYx4 ( Figure 61) was designed to express a direct repeat of four PVY sequences driven by the 35S promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx4 were cloned as a Xhol/BamHI fragment into xhol/BamHI cut pART7. Sequences consisting of the 35S promoter, direct repeats of PVY and the OCS terminator were excised as a NotI fragment from pART7.PVYx3 and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.PVYx3.
  • Plasmid pART7. PVY. LNYV. PVY ( Figure 62) was designed to express the interrupted direct repeat of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the interrupted direct repeat of PVY from 64 -
  • Plasmid pART7. PVY. LNYV. YVP ⁇ ( Figure 63) was designed to express the partial interrupted palindrome of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the partial interrupted palindrome of PVY sequences from pBC.PVY. LNYV. YVP ⁇ as a Xhol/Xbal fragment into pART7 digested with Xhol/Xbal. Sequences consisting of the 35S promoter, the partial interrupted palindrome of PVY sequences and the OCS terminator were excised from pART7. PVY. LNYV. YVP ⁇ as a NotI fragment and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27. PVY. LNYV. YVP.
  • Plasmid pART7. PVY. LNYV. YVP ( Figure 64) was designed to express the interrupted palindrome of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the interrupted palindrome of PVY sequences from PBC.PVY.LNYV.YVP ⁇ as a Xhol/Xbal fragment into pART7 digested with Xhol/Xbal. Sequences consisting of the 35S promoter, the interrupted palindrome of PVY sequences and the OCS terminator were excised from pART7.PVY.LNYV. YVP as a NotI fragment and cloned into ⁇ ART27, a plasmid with the desired insert orientation was selected and designated pART27. PVY. LNYV. YVP.
  • Plasmid pART7.35S. PVY. SCBV. YVP ( Figure 65) was designed to co-express sense and antisense constructs in transgenic plants. To generate this plasmid the PVY 65
  • Plasmid pART7.35S.PVYx3.SCBV.YVPx3 ( Figure 66) was designed to co-express sense and antisense repeats of PVY in transgenic plants, to generate this plasmid, the intermediate pART7.35S.O.SCBV.YVPx3 was constructed by cloning the triple direct PVY repeat from ClapBC.PVYx3 as a Clal/Accl fragment into Cla-digested p35S.SCBV.cass and isolating a plasmid with an antisense orientation, for p35S.PVYx3.SCBV.YVPx3 the triple direct PVY repeat from Cla pBC.PVYx3 was cloned as a KpnI/Smal fragment into KpnI/Smal-digested p35S.O.SCBV.YVPx3 to create p35S.PVYx3.SCBV.YVPx3.
  • Plasmids pART7.PVYx3.LNYV.YVPx3 & pART27.PVYx3.LNYV.YVPx3 Plasmid pART7.PVYx3.LNYV.YVPx3 ( Figure 67) was designed to express triple repeats of PVY sequences as an interrupted palindrome. To generate this plasmid an intermediate, pART7x3. PVY. LNYV. YV was constructed by cloning a PVY.LNYV.YVP fragment from pBC.PVY. LNYV. YVP as an Accl/Clal fragment into the plasmid pART7.PVYx2.
  • pART7.35S.PVYx3.LNYV.YVPx3 was made by cloning an additional PVY direct repeat from pBC.PVYx2 as an Accl/Clal fragment into Clal digested pART7x3.PVY.LNYV.YVP. Sequences from pART7.35S.PVYx3.LNYV.YVPx3, including the 35S promoter, all PVY sequences and the OCS terminator were excised as a NotI fragment and cloned into Notl-digested pART27, a plasmid with an - 66 -
  • Plasmid pART7.35S.PVY multi ( Figure 68) was designed to express higher order direct repeats of regions of PVY sequences in transgenic plants. Higher order direct repeats of a 72 bp of the PVY Nia region from PVY were prepared by annealing two partially complementary oligonucleotides as follows:
  • PVY1 5'-TAATGAGGATGATGTCCCTACCTTTAATTGGCAGAAATTTCTGTGGAAAGACAG GGAAATCTTTCGGCATTT-3';
  • PVY2 ⁇ '-TTCTGCCAATTAAAGGTAGGGACATCATCCTCATTAAAATGCCGAAAGATT TCCCTGTCTTTCCACAGAAAT-3'
  • oligonucleotides were phosphorylated with T4 polynucleotide kinase, heated and cooled slowly to permit self-annealing, ligated with T4 DNA ligase, end-filled with Klenow polymerase and cloned into pCR2.1 (Invitrogen). Plasmids containing multiple repeats were isolated and sequences were cloned as EcoRI fragments in a sense orientation into EcoRI-digested pART7, to create the intermediate pART7.PVY multi. to create pART27.PVY multi, the 35S promoter, PVY sequences and the OCS terminator were excised as a NotI fragment and cloned into Notl-digested pART27. A plasmid with an appropriate insert orientation was isolated and designated pART27. PVY multi. - 67 -
  • Viral immune lines are created by expressing viral sequences in stably transformed cell lines.
  • lytic viruses are used for this approach since cell lysis provides very simple screens and also offer the ability to directly select for potentially rare transformation events which might create viral immunity.
  • Sub-genomic fragments derived from a simple single stranded RNA virus (Bovine enterovirus - BEV) or a complex double stranded DNA virus, Herpes Simplex Virus I (HSV I) are cloned into a suitable vector and expressed in transformed cells.
  • Mammalian cell lines are transformed with genetic constructs designed to express viral sequences driven by the strong cytomegalovirus (CMV-IE) promoter. Sequences utilised include specific viral replicase genes. Random "shotgun" libraries comprising representative viral gene sequences, may also be used and the introduced dispersed nucleic acid molecule, to target the expression of virus sequences.
  • CMV-IE cytomegalovirus
  • Exemplary genetic constructs for use in this procedure comprising nucleotide sequences derived from the BEV RNA-dependent RNA polymerase gene, are presented herein.
  • Resistant cell lines are supportive of the ability of the introduced nucleotide sequences to inactivate viral gene expression in a mammalian system. - 68 -
  • resistant lines obtained from such experiments are used to more precisely define molecular and biochemical characteristics of the modulation which is observed.
  • PVYx3.SCBV.YPVx3, pART27.PVYx3.LNYV.YVPx3 and pART27.PVYx10 using tri-parental matings. DNA mini-preps from these strains were prepared and examined by restriction with NotI to ensure they contained the appropriate binary vectors.
  • Nicotiana tabaccum (cultivar W38) were transformed with these Agrobactehum strains using standard procedures. Putative transformed shoots were excised and rooted on media containing kanamycin. Under these conditions we have consistently observed that only transgenic shoots will root on kanamycin plates. Rooted shoots were transferred to soil and allowed to establish. After two to three weeks, vigorous plants with at least three sets of leaves were chosen and infected with PVY.
  • Viral inoculum was prepared from W38 tobacco previously infected with the virus, approximately 2 g of leaf material, showing obvious viral symptoms were ground with carbarundum in 10 ml of 100mM Na phosphate buffer (pH 7.5). the inoculum was diluted to 200 ml with additional Na phosphate buffer. Two leaves from each transgenic plant were sprinkled with carbarundum, then 0.4 ml of inoculum was applied to each leaf and leaves rubbed fairly vigorously with fingers. Using this procedure 100% of non-transgenic control plants were infected with PVY. 69
  • PVY-D an Australian PVY isolate
  • Transgenic lines were described as resistant if they showed reduced viral symptoms, which manifests as a reduction in the leaf are showing chlorotic lesions. Resistance ranges from very strong resistance where only a few viral lesions are observed on a plant to weak resistance which manifects as reduced symptoms on leaves that develop late in plant growth.
  • Transgenic plants which showed absolutely no evidence of viral symptoms were classified as immune. To ensure these plants were immune they were re-inoculated with virus, most plants remained immune, the few that showed symptoms were re- classified as resistant.
  • inverted and/or direct repeat sequences modulate expression of the virus target gene in the transgenic plant.
  • porcine PK2 cells were transformed with the relevant constructs.
  • PK2 cells constitutively express Gait enzyme, the activity of which results in the addition of a variety of ⁇ -1 ,3-galactosyl groups to a range of proteins expressed on the cell surface of these cells.
  • Cells were transformed using lipofectin and stably transformed lines were selected using genetecin.
  • HBSS/Hepes Hank's buffered saline solution with 20 mM Hepes, pH7.4
  • IB4-biotin 10 ug/ml IB4-biotin (Sigma) in HBSS/Hepes for 45 mins at 4°C.
  • Cells were washed in HBSS/Hepes, probed with a 1:200 dilution of ExtrAvidin-FITC (Sigma) in HBSS/Hepes for 45 mins at 4°C at and rinsed in cold HBSS/Hepes prior to FACS - 71 -
  • transformed cell lines are assayed for Gait inactivation and quantitative assessment of construct effectiveness is determined.
  • cell lines showing Gait inactivation are isolated and subject to further molecular analyses to determine the mechanism of gene inactivation.

Abstract

The present invention relates generally to a method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention utilises recombinant DNA technology to post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, organ or whole organism, thereby producing novel phenotypes. Novel synthetic genes and genetic constructs which are capable or repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.

Description

CONTROL OF GENE EXPRESSION
FIELD OF THE INVENTION
The present invention relates generally to a method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention utilises recombinant DNA technology to post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, organ or whole organism, thereby producing novel phenotypes. Novel synthetic genes and genetic constructs which are capable of repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.
GENERAL Bibliographic details of the publications referred to in this specification are collected at the end of the description.
As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular specified source or species, albeit not necessarily directly from that specified source or species.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
Sequence identity numbers (SEQ ID NOS.) containing nucleotide and amino acid sequence information included in this specification are collected after the Abstract and have been prepared using the programme Patentln Version 2.0. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1 , <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211 >, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1 , <400>2, etc).
The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue. The designation of amino acid residues referred to herein, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission, are listed in Table 1.
TABLE 1
Amino Acid Three-letter code One-letter code
Alanine Ala A
Arginine Arg R
Asparagine Asn N Aspartic acid Asp D
Cysteine Cys C
Glutamine Gin Q
Glutamic acid Glu E
Glycine Gly G Histidine His H
Isoleucine lie 1
Leucine Leu L
Lysine Lys K
Methionine Met M Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp w Tyrosine Tyr Y
Valine Val V
Aspartate/Asparagine Baa B
Glutamate/Glutamine Zaa z
Any amino acid Xaa X
Figure imgf000005_0001
BACKGROUND TO THE INVENTION
Controlling metabolic pathways in eukaryotic organisms is desirable for the purposes of producing novel traits therein or introducing novel traits into a particular cell, tissue or organ of said organism. Whilst recombinant DNA technology has provided significant progress in an understanding of the mechanisms regulating eukaryotic gene expression, much less progress has been made in the actual manipulation of gene expression to produce novel traits. Moreover, there are only limited means by which human intervention may lead to a modulation of the level of eukaryotic gene expression.
One approach to repressing, delaying or otherwise reducing gene expression utilise a mRNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed and capable of being translated into a polypeptide. Although the precise mechanism involved in this approach is not established, it has been postulated that a double-stranded mRNA may form by base pairing between the complementary nucleotide sequences, to produce a complex which is translated at low efficiency and/or degraded by intracellular ribonuclease enzymes prior to being translated.
Alternatively, the expression of an endogenous gene in a cell, tissue or organ may be suppressed when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell. Whilst the mechanism involved in this phenomenon has not been established and appears to be involve mechanistically heterogeneous processes. For example, this approach has been postulated to involve transcriptional repression, in which case somatically-heritable repressed states of chromatin are formed or alternatively, a post-transcriptional silencing wherein transcription initiation occurs normally but the RNA products of the co-suppressed genes are subsequently eliminated.
The efficiency of both of these approaches in targeting the expression of specific genes is very low and highly variable results are usually obtained. Inconsistent results are obtained using different regions of genes, for example 5'- untranslated regions, 3'-untranslated regions, coding regions or intron sequences to target gene expression. Accordingly, there currently exists no consensus as to the nature of genetic sequences which provide the most efficient means for repressing, delaying or otherwise reducing gene expression using existing technologies. Moreover, such a high degree of variation exists between generations such that it is not possible to predict the level of repression of a specific gene in the progeny of an organism in which gene expression was markedly modified.
Recently, Dorer and Henikoff (1994) demonstrated the silencing of tandemly repeated gene copies in the Drosophila genome and the transcriptional repression of dispersed Drosophila Adh genes by Polycomb genes (i.e. the Pc-G system; Pal-Bhadra et al, 1997). However, such silencing of tandemly repeated gene copies is of little utility in an attempt to manipulate gene expression in an animal cell by recombinant means, wherein the sequences capable of targeting the expression of a particular gene are introduced at dispersed locations in the genome, absent the combination of this approach with gene-targeting technology. Whilst theoretically possible, such combinations would be expected to work at only low-efficiency, based upon the low efficiency of gene-targeting approaches used in isolation and further, would require complicated vector systems. Additionally, the utilisation of transcriptional repression, such as the Drosophila Pc-G system, would appear to require some knowledge of the regulatory mechanisms capable of modulating the expression of any specific target gene and, as a consequence, would be difficult to implement in practice as a general technology for repressing, delaying or reducing gene expression in animal cells.
The poor understanding of the mechanisms involved in these phenomena has meant that there have been few improvements in technologies for modulating the level of gene expression , in particular technologies for delaying, repressing or otherwise reducing the expression of specific genes using recombinant DNA technology. - 6
Furthermore, as a consequence of the unpredictability of these approaches, there is currently no commercially-viable means for modulating the level of expression of a specific gene in a eukaryotic or prokaryotic organism.
Thus, there exists a need for improved methods of modulating gene expression, in particular repressing, delaying or otherwise reducing gene expression in animal cells for the purpose of introducing novel phenotypic traits thereto. In particular, these methods should provide general means for phenotypic modification, without the necessity for performing concomitant gene-targeting approaches.
SUMMARY OF THE INVENTION
The invention is based in part on the surprising discovery by the inventors that cells which exhibit one or more desired traits can be produced and selected from transformed cells comprising a nucleic acid molecule operably linked to a promoter, wherein the transcription product of the nucleic acid molecule comprises a nucleotide sequence which is substantially identical to the nucleotide sequence of a transcript of an endogenous or non-endogenous target gene, the expression of which is intended to be modulated. The transformed cells are regenerated into whole tissues, organs or organisms capable of exhibiting novel traits, in particular virus resistance and modified expression of endogenous genes.
Accordingly, one aspect of the present invention provides a method of modulating the expression of a target gene in an animal cell, tissue or organ, said method at least comprising the step of introducing to said cell, tissue or organ one or more dispersed nucleic acid molecules or foreign nucleic acid molecules comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or complementary thereto for a time and under conditions sufficient for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced. - 7
In a particularly preferred embodiment, the dispersed nucleic acid molecules or foreign nucleic acid molecules comprises a nucleotide sequence which encodes multiple copies of an mRNA molecule which is substantially identical to the nucleotide sequence of the mRNA product of the target gene. More preferably, the multiple copies of the target molecule are tandem direct repeat sequences.
In a more particularly preferred embodiment, the dispersed nucleic acid molecule or foreign nucleic acid molecule is in an expressible form such that it is at least capable of being transcribed to produce mRNA.
The target gene may be a gene which is endogenous to the animal cell or alternatively, a foreign gene such as a viral or foreign genetic sequence, amongst others. Preferably, the target gene is a viral genetic sequence.
The invention is particularly useful in the modulation of eukaryotic gene expression, in particular the modulation of human or animal gene expression and even more particularly in the modulation of expression of genes derived from vertebrate and invertebrate animals, such as insects, aquatic animals (eg. fish, shellfish, molluscs, crustaceans such as crabs, lobsters and prawns, avian animals and mammals, amongst others).
A variety of traits are selectable with appropriate procedures and sufficient numbers of transformed cells. Such traits include, but are not limited to, visible traits, disease- resistance traits, and pathogen-resistance traits. The modulatory effect is applicable to a variety of genes expressed in plants and animals including, for example, endogenous genes responsible for cellular metabolism or cellular transformation, including oncogenes, transcription factors and other genes which encode polypeptides involved in cellular metabolism.
For example, an alteration in the pigment production in mice can be engineered by - 8
targeting the expression of the tyrosinase gene therein. This provides a novel phenotype of albinism in black mice. By targeting genes required for virus replication in a plant cell or an animal cell, a genetic construct which comprises multiple copies of nucleotide sequence encoding a viral replicase, polymerase, coat protein or uncoating gene, or protease protein, may be introduced into a cell where it is expressed, to confer immunity against the virus upon the cell.
In performance of the present invention, the dispersed nucleic acid molecule or foreign nucleic acid molecule will generally comprise a nucleotide sequence having greater than about 85% identity to the target gene sequence, however, a higher homology might produce a more effective modulation of expression of the target gene sequence. Substantially greater homology, or more than about 90% is preferred, and even more preferably about 95% to absolute identity is desirable.
The introduced dispersed nucleic acid molecule or foreign nucleic acid molecule sequence, needing less than absolute homology, also need not be full length, relative to either the primary transcription product or fully processed mRNA of the target gene. A higher homology in a shorter than full length sequence compensates for a longer less homologous sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective. Normally, a sequence of greater than 20-100 nucleotides should be used, though a sequence of greater than about 200-300 nucleotides would be preferred, and a sequence of greater than 500-1000 nucleotides would be especially preferred depending on the size of the target gene.
A second aspect of the present invention provides a synthetic gene which is capable of modifying target gene expression in a cell, tissue or organ of a prokaryotic or eukaryotic organism which is transfected or transformed therewith, wherein said synthetic gene at least comprises a dispersed nucleic acid molecular foreign nucleic acid molecule comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a derivative thereof or a complementary sequence thereto placed operably under the control of a promoter sequence which is operable in said cell, tissue or organ.
A third aspect of the invention provides a synthetic gene which is capable of modifying the expression of a target gene in a cell, tissue or organ of a prokaryotic or eukaryotic organism which is transfected or transformed therewith, wherein said synthetic gene at least comprises multiple structural gene sequences, wherein each of said structural gene sequences comprises a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a derivative thereof or a complementary sequence thereto and wherein said multiple structural gene sequences are placed operably under the control of a single promoter sequence which is operable in said cell, tissue or organ.
A fourth aspect of the present invention provides a synthetic gene which is capable of modifying the expression of a target gene in a cell, tissue or organ of a prokaryote or eukaryote which is transfected or transformed therewith wherein said synthetic gene at least comprises multiple structural gene sequences wherein each of said structural gene sequences is placed operably under the control of a promoter sequence which is operable in said cell, tissue or organ and wherein each of said structural gene sequences comprises a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a derivative thereof or a complementary sequence thereto.
A fifth aspect of the present invention provides a genetic construct which is capable of modifying the expression of an endogenous gene or target gene in a transformed or transfected cell, tissue or organ wherein said genetic construct at least comprises the synthetic gene of the invention and one or more origins of replication and/or selectable marker gene sequences. - 10 -
ln order to observe many novel traits in multicellular organisms such as plants and animals, in particular those which are tissue-specific or organ-specific or developmentally-regulated, regeneration of a transformed cell carrying the synthetic genes and genetic constructs described herein into a whole organism will be required. Those skilled in the art will be aware that this means growing a whole organism from a transformed plant cell or animal cell, a group of such cells, a tissue or organ. Standard methods for the regeneration of certain plants and animals from isolated cells and tissues are known to those skilled in the art.
Accordingly, a sixth aspect of the invention provides a cell, tissue, organ or organism comprising the synthetic genes and genetic constructs described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a diagrammatic representation of the plasmid pEGFP-N1 MCS.
Figure 2 is a diagrammatic representation of the plasmid pCMV.cass.
Figure 3 is a diagrammatic representation of the plasmid pCMV.SV40L.cass.
Figure 4 is a diagrammatic representation of the plasmid pCMV.SV40LR.cass.
Figure 5 is a diagrammatic representation of the plasmid pCR.Bgl-GFP-Bam.
Figure 6 is a diagrammatic representation of the plasmid pBSII(SK+).EGFP.
Figure 7 is a diagrammatic representation of the plasmid pCMV.EGFP.
Figure 8 is a diagrammatic representation of the plasmid pCR.SV40L. - 11
Figure 9 is a diagrammatic representation of the plasmid pCR.BEVJ .
Figure 10 is a diagramma ic representation of the plasmid pCR.BEV.2.
Figure 11 is a diagramma' ic representation of the plasmid pCR.BEV.3.
Figure 12 is a diagramma ic representation of the plasmid pCMV.EGFP.BEV2.
Figure 13 is a diagramma ic representation of the plasmid pCMV.BEV.2.
Figure 14 is a diagramma ic representation of the plasmid pCMV.BEV.3.
Figure 15 is a diagramma ic representation of the plasmid pCMV.VEB.
Figure 16 is a diagramma ic representation of the plasmid pCMV.BEV.GFP.
Figure 17 is a diagramma ic representation of the plasmid pCMV.BEV.SV40L-0.
Figure 18 is a diagramma ic representation of the plasmid pCMV.0.SV40L.BEV.
Figure 19 is a diagramma ic representation of the plasmid pCMV.0.SV40L.VEB.
Figure 20 is a diagramma ic representation of the plasmid pCMV.BEVx2.
Figure 21 is a diagramma ic representation of the plasmid pCMV.BEVx3.
Figure 22 is a diagramma ic representation of the plasmid pCMV.BEVx4.
Figure 23 is a diagramma ic representation of the plasmid pCMV.BEV.SV40L.BEV. - 12 -
Figure 24 is a diagrammatic representation of the plasmid pCMV.BEV.SV40L.VEB.
Figure 25 is a diagrammatic representation of the plasmid pCMV.BEV.GFP.VEB.
Figure 26 is a diagrammatic representation of the plasmid pCMV.EGFP.BEV2.PFG.
Figure 27 is a diagrammatic representation of the plasmid pCMV.BEV.SV40LR.
Figure 28 is a diagrammatic representation of the plasmid pCDNA3.Galt.
Figure 29 is a diagrammatic representation of the plasmid pCMV.Galt.
Figure 30 is a diagrammatic representation of the plasmid pCMV.EGFP.Galt.
Figure 31 is a diagrammatic representation of the plasmid pCMV.Galt.GFP.
Figure 32 is a diagrammatic representation of the plasmid pCMV.Galt.SV40L.0.
Figure 33 is a diagrammatic representation of the plasmid pCMV.Galt.SV40L.tlaG.
Figure 34 is a diagrammatic representation of the plasmid pCMV.0.SV40L.Galt.
Figure 35 is a diagrammatic representation of the plasmid pCMV.Galtx2.
Figure 36 is a diagrammatic representation of the plasmid pCMV.Galtx4.
Figure 37 is a diagrammatic representation of the plasmid pCMV. Gait. SV40L. Gait.
Figure 38 is a diagrammatic representation of the plasmid pCMV.Galt. SV40L.tlaG. 13
Figure 39 is a diagramma ic representation of the plasmid pCMV.Galt.GFP.tlaG.
Figure 40 is a diagramma' ic representation of the plasmid pCMV.EGFP.Galt.PFG.
Figure 41 is a diagramma ic representation of the plasmid pCMV.Galt.SV40LR.
Figure 42 is a diagramma ic representation of the plasmid pART7.
Figure 43 is a diagramma c representation of the plasmid pART7.35S.SCBV.cass.
Figure 44 is a diagramma ic representation of the plasmid pBC.PVY.
Figure 45 is a diagramma ic representation of the plasmid pSP72.PVY.
Figure 46 is a diagramma ic representation of the plasmid pClapBC.PVY.
Figure 47 is a diagramma ic representation of the plasmid pBC.PVYx2.
Figure 48 is a diagramma ic representation of the plasmid pSP72.PVYx2.
Figure 49 is a diagramma ic representation of the plasmid pBC.PVYx3.
Figure 50 is a diagramma ic representation of the plasmid pBC.PVYx4.
Figure 51 is a diagramma ic representation of the plasmid pBC.PVY.LNYV.
Figure 52 is a diagramma' ic representation of the plasmid pBC.PVY.LNYV.PVY.
Figure 53 is a diagramma ic representation of the plasmid pBC.PVY.LNYV. YVPΔ. - 14 -
Figure 54 is a diagrammatic representation of the plasmid pBC.PVY.LNYV.YVP.
Figure 55 is a diagrammatic representation of the plasmid pART27.PVY
Figure 56 is a diagrammatic representation of the plasmid pART27.35S.PVY.SCBV.O.
Figure 57 is a diagrammatic representation of the plasmid pART27.35S.O.SCBV.PVY.
Figure 58 is a diagrammatic representation of the plasmid pART27.35S.O.SCBV.YVP.
Figure 59 is a diagrammatic representation of the plasmid pART7.PVYx2.
Figure 60 is a diagrammatic representation of the plasmid pART7.PVYx3.
Figure 61 is a diagrammatic representation of the plasmid pART7.PVYx4.
Figure 62 is a diagrammatic representation of the plasmid pART7.PVY.LNYV.PVY.
Figure 63 is a diagrammatic representation of the plasmid pART7.PVY.LNYV. YVPΔ.
Figure 64 is a diagrammatic representation of the plasmid pART7.PVY.LNYV.YVP.
Figure 65 is a diagrammatic representation of pART7.35S.PVY.SCBV.YVP.
Figure 66 is a diagrammatic representation of pART7.35S.PVYx3.SCBV.YVPx3.
Figure 67 is a diagrammatic representation of pART7.PVYx3.LNYV.YVPx3.
Figure 68 is a diagrammatic representation of the plasmid pART7.PVYMULTI. 15
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of modulating the expression of a target gene in a cell, tissue or organ, said method at least comprising the step of introducing to said cell, tissue or organ one or more dispersed nucleic acid molecules or foreign nucleic acid molecules comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or complementary thereto for a time and under conditions sufficient for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
By "multiple copies" is meant that two or more copies of the target gene are presented in close physical connection or juxtaposed, in the same or different orientation, on the same nucleic acid molecule, optionally separated by a stuffer fragment or intergenic region to facilitate secondary structure formation between each repeat where this is required. The stuffer fragment may comprise any combination of nucleotide or amino acid residues, carbohydrate molecules or oligosaccharide molecules or carbon atoms or a homologue, analogue or derivative thereof which is capable of being linked covalently to a nucleic acid molecule.
Preferably, embodiment, the stuffer fragment comprises a sequence of nucleotides or a homologue, analogue or derivative thereof.
More preferably, the stuffer fragment comprises a sequence of nucleotides of at least about 10-50 nucleotides in length, even more preferably at least about 50-100 nucleotides in length and still more preferably at least about 100-500 nucleotides in length.
Wherein the dispersed or foreign nucleic acid molecule comprises intron/exon splice 16 -
junction sequences, the stuffer fragment may serve as an intron sequence placed between the 3'-splice site of the structural gene nearer the 5'-end of the gene and the 5'- splice site of the next downstream unit thereof. Alternatively, wherein it is desirable for more than two adjacent nucleotide sequence units of the dispersed foreign nucleic acid molecule to be translated, the stuffer fragment placed there between should not include an in-frame translation stop codon, absent intron/exon splice junction sequences at both ends of the stuffer fragment or the addition of a translation start codon at the 5' end of each unit, as will be obvious to those skilled in the art.
Preferred stuffer fragments are those which encode detectable marker proteins or biologically-active analogues and derivatives thereof, for example luciferase, β- galacturonase, β-galactosidase, chloramphenicol acetyltransferase or green fluorescent protein, amongst others. Additional stuffer fragments are not excluded.
According to this embodiment, the detectable marker or an analogue or derivative thereof serves to indicate the expression of the synthetic gene of the invention in a cell, tissue or organ by virtue of its ability to confer a specific detectable phenotype thereon, preferably a visually-detectable phenotype.
As used herein, the term "modulating" shall be taken to mean that expression of the target gene is reduced in amplitude and/or the timing of gene expression is delayed and/or the developmental or tissue-specific or cell-specific pattern of target gene expression is altered, compared to the expression of said gene in the absence of the inventive method described herein.
Whilst not limiting the scope of the invention described herein, the present invention is directed to a modulation of gene expression which comprises the repression, delay or reduction in amplitude of target gene expression in a specified cell, tissue or organ of a eukaryotic organism, in particular a plant such as a monocotyledonous or dicotyledonous plant, or a human or other animal and even more particularly a 17
vertebrate and invertebrate animal, such as an insect, aquatic animal (eg. fish, shellfish, mollusc, crustacean such as a crab, lobster or prawn, an avian animal or a mammal, amongst others).
More preferably, target gene expression is completely inactivated by the dispersed nucleic acid molecules or foreign nucleic acid molecules which has been introduced to the cell, tissue or organ.
Whilst not being bound by any theory or mode of action, the reduced or eliminated expression of the target gene which results from the performance of the invention may be attributed to reduced or delayed translation of the mRNA transcription product of the target gene or alternatively, the prevention of translation of said mRNA, as a consequence of sequence-specific degradation of the mRNA transcript of the target gene by an endogenous host cell system.
It is particularly preferred that, for optimum results, sequence-specific degradation of the mRNA transcript of the target gene occurs either prior to the time or stage when the mRNA transcript of the target gene would normally be translated or alternatively, at the same time as the mRNA transcript of the target gene would normally be translated. Accordingly, the selection of an appropriate promoter sequence to regulate expression of the introduced dispersed nucleic acid molecule or foreign nucleic acid molecule is an important consideration to optimum performance of the invention. For this reason, strong constitutive promoters or inducible promoter systems are especially preferred for use in regulating expression of the introduced dispersed nucleic acid molecules or foreign nucleic acid molecules.
The present invention clearly encompasses reduced expression wherein reduced expression of the target gene is effected by lowered transcription, subject to the proviso that a reduction in transcription is not the sole mechanism by which this occurs and said reduction in transcription is at least accompanied by reduced translation of 18
the steady-state mRNA pool.
The target gene may be a genetic sequence which is endogenous to the animal cell or alternatively, a non-endogenous genetic sequence, such as a genetic sequence which is derived from a virus or other foreign pathogenic organism and is capable of entering a cell and using the cell's machinery following infection.
Wherein the target gene is a non-endogenous genetic sequence to the animal cell, it is desirable that the target gene encodes a function which is essential for replication or reproduction of the viral or other pathogen. In such embodiments, the present invention is particularly useful in the prophylactic and therapeutic treatment of viral infection of an animal cell or for conferring or stimulating resistance against said pathogen.
Preferably, the target gene comprises one or more nucleotide sequences of a viral pathogen of a plant or an animal cell, tissue or organ.
For example, in the case of animals and humans, the viral pathogen may be a retrovirus, for example a lentivirus such as the immunodeficiency viruses, a single- stranded (+) RNA virus such as bovine enterovirus (BEV) or Sinbis alphavirus. Alternatively, the target gene can comprise one or more nucleotide sequences of a viral pathogen of an animal cell, tissue or organ, such as but not limited to a double- stranded DNA virus such as bovine herpes virus or herpes simplex virus I (HSV I), amongst others.
In the case of plants, the virus pathogen is preferably a potyvirus, caulimovirus, badnavirus, geminivirus, reovirus, rhabdovirus, bunyavirus, tospovirus, tenuivirus, tombusvirus, luteovirus, sobemovirus, bromovirus, cucomovirus, ilavirus, alfamovirus, tobamovirus, tobravirus, potexvirus and clostrovirus, such as but not limited to CaMV, SCSV, PVX, PVY, PLRV, and TMV, amongst others. - 19 -
With particular regard to viral pathogens, those skilled in the art are aware that virus- encoded functions may be complemented in trans by polypeptides encoded by the host cell. For example, the replication of the bovine herpes virus genome in the host cell may be facilitated by host cell DNA polymerases which are capable of complementing an inactivated viral DNA polymerase gene.
Accordingly, wherein the target gene is a non-endogenous genetic sequence to the animal cell, a further alternative embodiment of the invention provides for the target gene to encode a viral or foreign polypeptide which is not capable of being complemented by a host cell function, such as a virus-specific genetic sequence. Exemplary target genes according to this embodiment of the invention include, but are not limited to genes which encode virus coat proteins, uncoating proteins and RNA- dependent DNA polymerases and RNA-dependent RNA polymerases, amongst others.
In a particularly preferred embodiment of the present invention, the target gene is the BEV RNA-dependent RNA polymerase gene or a homologue, analogue or derivative thereof or PVY Nia protease-encoding sequences.
The cell in which expression of the target gene is modified may be any cell which is derived from a multicellular plant or animal, including cell and tissue cultures thereof. Preferably, the animal cell is derived from an insect, reptile, amphibian, bird, human or other mammal. Exemplary animal cells include embryonic stem cells, cultured skin fibroblasts, neuronal cells, somatic cells, haematopoietic stem cells, T-cells and immortalised cell lines such as COS, VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, baby hamster kidney (BHK) or MDBK cell lines, amongst others. Such cells and cell lines are readily available to those skilled in the art. Accordingly, the tissue or organ in which expression of the target gene is modified may be any tissue or organ comprising such animal cells.
Preferably the plant cell is derived from a monocotyledonous or dicotyledonous plant 20
species or a cell line derived therefrom.
As used herein, the term "dispersed nucleic acid molecule" shall be taken to refer to a nucleic acid molecule which comprises one or more multiple copies, preferably tandem direct repeats, of a nucleotide sequence which is substantially identical or complementary to the nucleotide sequence of a gene which originates from the cell, tissue or organ into which said nucleic acid molecule is introduced, wherein said nucleic acid molecule is non-endogenous in the sense that it is introduced to the cell, tissue or organ of an animal via recombinant means and will generally be present as extrachromosomal nucleic acid or alternatively, as integrated chromosomal nucleic acid which is genetically-unlinked to said gene. More particularly, the "dispersed nucleic acid molecule" will comprise chromosomal or extrachromosomal nucleic acid which is unlinked to the target gene against which it is directed in a physical map, by virtue of their not being tandemly-linked or alternatively, occupying a different chromosomal position on the same chromosome or being localised on a different chromosome or present in the cell as an episome, plasmid, cosmid or virus particle.
By "foreign nucleic acid molecule" is meant an isolated nucleic acid molecule which has one or more multiple copies, preferably tandem direct repeats, of a nucleotide sequence which originates from the genetic sequence of an organism which is different from the organism to which the foreign nucleic acid molecule is introduced. This definition encompasses a nucleic acid molecule which originates from a different individual of the same lowest taxonomic grouping (i.e. the same population) as the taxonomic grouping to which said nucleic acid molecule is introduced, as well as a nucleic acid molecule which originates from a different individual of a different taxonomic grouping as the taxonomic grouping to which said nucleic acid molecule is introduced, such as a gene derived from a viral pathogen.
Accordingly, a target gene against which a foreign nucleic acid molecule acts in the performance of the invention may be a nucleic acid molecule which has been 21
introduced from one organism to another organism using transformation or introgression technologies. Exemplary target genes according to this embodiment of the invention include the green fluorescent protein-encoding gene derived from the jellyfish Aequoria victoria (Prasher et a/., 1992; International Patent Publication No. WO 95/07463), tyrosinase genes and in particular the murine tyrosinase gene (Kwon et al., 1988), the Escherichia coli lac\ gene which is capable of encoding a polypeptide repressor of the lacZ gene, the porcine α-1 ,3-galactosyltransferase gene (NCBI Accession No. L36535) exemplified herein, and the PVY and BEV structural genes exemplified herein or a homologue, analogue or derivative of said genes or a complementary nucleotide sequence thereto.
The present invention is further useful for simultaneously targeting the expression of several target genes which are co-expressed in a particular cell, for example by using a dispersed nucleic acid molecule or foreign nucleic acid molecule which comprises nucleotide sequences which are substantially identical to each of said co-expressed target genes.
By "substantially identical" is meant that the introduced dispersed or foreign nucleic acid molecule of the invention and the target gene sequence are sufficiently identical at the nucleotide sequence level to permit base-pairing there between under standard intracellular conditions.
Preferably, the nucleotide sequence of each repeat in the dispersed or foreign nucleic acid molecule of the invention and the nucleotide sequence of a part of the target gene sequence are at least about 80-85% identical at the nucleotide sequence level, more preferably at least about 85-90% identical, even more preferably at least about 90-95% identical and still even more preferably at least about 95-99% or 100% identical at the nucleotide sequence level.
Notwithstanding that the present invention is not limited by the precise number of 22 -
repeated sequences in the dispersed nucleic acid molecule or the foreign nucleic acid molecule of the invention, it is to be understood that the present invention requires at least two copies of the target gene sequence to be expressed in the cell.
Preferably, the multiple copies of the target gene sequence are presented in the dispersed nucleic acid molecule or the foreign nucleic acid molecule as tandem inverted repeat sequences and/or tandem direct repeat sequences. Such configurations are exemplified by the "test plasmids" described herein that comprise Gait, BEV or PVY gene regions.
Preferably, the dispersed or foreign nucleic acid molecule which is introduced to the cell, tissue or organ comprises RNA or DNA.
Preferably, the dispersed or foreign nucleic acid molecule further comprises a nucleotide sequence or is complementary to a nucleotide sequence which is capable of encoding an amino acid sequence encoded by the target gene. Even more preferably, the nucleic acid molecule includes one or more ATG or AUG translational start codons.
Standard methods may be used to introduce the dispersed nucleic acid molecule or foreign nucleic acid molecule into the cell, tissue or organ for the purposes of modulating the expression of the target gene. For example, the nucleic acid molecule may be introduced as naked DNA or RNA, optionally encapsulated in a liposome, in a virus particle as attenuated virus or associated with a virus coat or a transport protein or inert carrier such as gold or as a recombinant viral vector or bacterial vector or as a genetic construct, amongst others.
Administration means include injection and oral ingestion (e.g. in medicated food material), amongst others. - 23
The subject nucleic acid molecules may also be delivered by a live delivery system such as using a bacterial expression system optimised for their expression in bacteria which can be incorporated into gut flora. Alternatively, a viral expression system can be employed. In this regard, one form of viral expression is the administration of a live vector generally by spray, feed or water where an infecting effective amount of the live vector (e.g. virus or bacterium) is provided to the animal. Another form of viral expression system is a non-replicating virus vector which is capable of infecting a cell but not replicating therein. The non-replicating viral vector provides a means of introducing to the human or animal subject genetic material for transient expression therein. The mode of administering such a vector is the same as a live viral vector.
The carriers, excipients and/or diluents utilised in delivering the subject nucleic acid molecules to a host cell should be acceptable for human or veterinary applications. Such carriers, excipients and/or diluents are well-known to those skilled in the art. Carriers and/or diluents suitable for veterinary use include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the composition is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
In an alternative embodiment, the invention provides a method of modulating the expression of a target gene in a cell, tissue or organ, said method at least comprising the steps of: (i) selecting one or more dispersed nucleic acid molecules or foreign nucleic acid molecules which comprise multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or which is complementary thereto; and
(ii) introducing said dispersed nucleic acid molecules or foreign nucleic acid molecules to said cell, tissue or organ for a time and under conditions sufficient - 24 -
for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
To select appropriate nucleotide sequences for targeting expression of the target gene, several approaches may be employed. In one embodiment, multiple copies of specific regions of characterised genes may be cloned in operable connection with a suitable promoter and assayed for efficacy in reducing target gene expression. Alternatively, shotgun libraries comprising multiple copies of genetic sequences may be produced and assayed for their efficacy in reducing target gene expression. The advantage associated with the latter approach is that it is not necessary to have any prior knowledge of the significance of any particular target gene in specifying an undesirable phenotype in the cell. For example, shotgun libraries comprising virus sub-genomic fragments may be employed and tested directly for their ability to confer virus immunity on the animal host cell, without prior knowledge of the role which any virus genes play in pathogenesis of the host cell.
As used herein, the term "shotgun library" is a set of diverse nucleotide sequences wherein each member of said set is preferably contained within a suitable plasmid, cosmid, bacteriophage or virus vector molecule which is suitable for maintenance and/or replication in a cellular host. The term "shotgun library" includes a representative library, in which the extent of diversity between the nucleotide sequences is numerous such that all sequences in the genome of the organism from which said nucleotide sequences is derived are present in the "set" or alternatively, a limited library in which there is a lesser degree of diversity between said sequences. The term "shotgun library" further encompasses random nucleotide sequences, wherein the nucleotide sequence comprises viral or cellular genome fragments, amongst others obtained for example by shearing or partial digestion of genomic DNA using restriction endonucleases, amongst other approaches. A "shotgun library" further includes cells, virus particles and bacteriophage particles comprising the - 25 -
individual nucleotide sequences of the diverse set.
Preferred shotgun libraries according to this embodiment of the invention are "representative libraries", comprising a set of tandem repeated nucleotide sequences derived from a viral pathogen of a plant or an animal.
In a particularly preferred embodiment of the invention, the shotgun library comprises cells, virus particles or bacteriophage particles comprising a diverse set of tandem- repeated nucleotide sequences which encode a diverse set of amino acid sequences, wherein the member of said diverse set of nucleotide sequences are placed operably under the control of a promoter sequence which is capable of directing the expression of said tandem-repeated nucleotide sequence in the cell.
Accordingly, the nucleotide sequence of each unit in the tandem-repeated sequence may comprise at least about 1 to 200 nucleotides in length. The use of larger fragments, particularly employing randomly sheared nucleic acid derived from viral, plant or animal genomes, is not excluded.
The introduced nucleic acid molecule is preferably in an expressible form.
By "expressible form" is meant that the subject nucleic acid molecule is presented in an arrangement such that it may be expressed in the cell, tissue, organ or whole organism, at least at the transcriptional level (i.e. it is expressed in the animal cell to yield at least an mRNA product which is optionally translatable or translated to produce a recombinant peptide, oligopeptide or polypeptide molecule).
By way of exemplification, in order to obtain expression of the dispersed nucleic acid molecule or foreign nucleic acid molecule in the cell, tissue or organ of interest, a synthetic gene or a genetic construct comprising said synthetic gene is produced, wherein said synthetic gene comprises a nucleotide sequence as described supra in 26
operable connection with a promoter sequence which is capable of regulating expression therein. Thus, the subject nucleic acid molecule will be operably connected to one or more regulatory elements sufficient for eukaryotic transcription to occur.
Accordingly, a further alternative embodiment of the invention provides a method of modulating the expression of a target gene in an animal cell, tissue or organ, said method at least comprising the steps of:
(i) selecting one or more dispersed nucleic acid molecules or foreign nucleic acid molecules which comprise multiple copies, preferably tandem repeats, of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or which is complementary thereto;
(ii) producing a synthetic gene comprising said dispersed nucleic acid molecules or foreign nucleic acid molecules; (iii) introducing said synthetic gene to said cell, tissue or organ; and
(iv) expressing said synthetic gene in said cell, tissue or organ for a time and under conditions sufficient for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
Reference herein to a "gene" or "genes" is to be taken in its broadest context and includes:
(i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); and/or
(ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5'- and 3'- untranslated sequences of the gene; and/or
(iii) a structural region corresponding to the coding regions (i.e. exons) optionally further comprising untranslated sequences and/or a heterologous promoter sequence which consists of transcriptional and/or translational regulatory regions capable of 27
conferring expression characteristics on said structural region.
The term "gene" is also used to describe synthetic or fusion molecules encoding all or part of a functional product, in particular a sense or antisense mRNA product or a peptide, oligopeptide or polypeptide or a biologically-active protein.
The term "synthetic gene" refers to a non-naturally occurring gene as hereinbefore defined which preferably comprises at least one or more transcriptional and/or translational regulatory sequences operably linked to a structural gene sequence.
The term "structural gene" shall be taken to refer to a nucleotide sequence which is capable of being transmitted to produce mRNA and optionally, encodes a peptide, oligopeptide, polypeptide or biologically active protein molecule. Those skilled in the art will be aware that not all mRNA is capable of being translated into a peptide, oligopeptide, polypeptide or protein, for example if the mRNA lacks a functional translation start signal or alternatively, if the mRNA is antisense mRNA. The present invention clearly encompasses synthetic genes comprising nucleotide sequences which are not capable of encoding peptides, oligopeptides, polypeptides or biologically-active proteins. In particular, the present inventors have found that such synthetic genes may be advantageous in modifying target gene expression in cells, tissues or organs of a prokaryotic or eukaryotic organism.
The term "structural gene region" refers to that part of a synthetic gene which comprises a dispersed nucleic acid molecule or foreign nucleic acid molecule as described herein which is expressed in a cell, tissue or organ under the control of a promoter sequence to which it is operably connected. A structural gene region may comprise one or more dispersed nucleic acid molecules and/or foreign nucleic acid molecules operably under the control of a single promoter sequence or multiple promoter sequences. Accordingly, the structural gene region of a synthetic gene may comprise a nucleotide sequence which is capable of encoding an amino acid 28 -
sequence or is complementary thereto. In this regard, a structural gene region which is used in the performance of the instant invention may also comprise a nucleotide sequence which encodes an animo acid sequence yet lacks a functional translation initiation codon and/or a functional translation stop codon and, as a consequence, does not comprise a complete open reading frame. In the present context, the term "structural gene region" also extends to a non-coding nucleotide sequences, such as 5'- upstream or 3'-downstream sequences of a gene which would not normally be translated in a eukaryotic cell which expresses said gene.
Accordingly, in the context of the present invention, a structural gene region may also comprise a fusion between two or more open reading frames of the same or different genes. In such embodiments, the invention may be used to modulate the expression of one gene, by targeting different non-contiguous regions thereof or alternatively, to simultaneously modulate the expression of several different genes, including different genes of a multigene family. In the case of a fusion nucleic acid molecule which is non- endogenous to the animal cell and in particular comprises two or more nucleotide sequences derived from a viral pathogen, the fusion may provide the added advantage of conferring simultaneous immunity or protection against several pathogens, by targeting the expression of genes in said several pathogens. Alternatively or in addition, the fusion may provide more effective immunity against any pathogen by targeting the expression of more than one gene of that pathogen.
Particularly preferred structural gene regions according to this aspect of the invention are those which include at least one translatable open reading frame, more preferably further including a translational start codon located at the 5'-end of said open reading frame, albeit not necessarily at the 5'-terminus of said structural gene region. In this regard, notwithstanding that the structural gene region may comprise at least one translatable open reading frame and/or AUG or ATG translational start codon, the inclusion of such sequences in no way suggests that the present invention requires translation of the introduced nucleic acid molecule to occur in order to modulate the - 29 -
expression of the target gene. Whilst not being bound by any theory or mode of action, the inclusion of at least one translatable open reading frame and/or translational start codon in the subject nucleic acid molecule may serve to increase stability of the mRNA transcription product thereof, thereby improving the efficiency of the invention.
The optimum number of structural gene sequences which may be involved in the synthetic gene of the present invention will vary considerably, depending upon the length of each of said structural gene sequences, their orientation and degree of identity to each other. For example, those skilled in the art will be aware of the inherent instability of palindromic nucleotide sequences in vivo and the difficulties associated with constructing long synthetic genes comprising inverted repeated nucleotide sequences, because of the tendency for such sequences to recombine in vivo. Notwithstanding such difficulties, the optimum number of structural gene sequences to be included in the synthetic genes of the present invention may be determined empirically by those skilled in the art, without any undue experimentation and by following standard procedures such as the construction of the synthetic gene of the invention using recombinase-deficient cell lines, reducing the number of repeated sequences to a level which eliminates or minimises recombination events and by keeping the total length of the multiple structural gene sequence to an acceptable limit, preferably no more than 5-1 Okb, more preferably no more than 2-5kb and even more preferably no more than 0.5-2. Okb in length.
Wherein the structural gene region comprises more than one dispersed nucleic acid molecule or foreign nucleic acid molecule it shall be referred to herein as a "multiple structural gene region" or similar term. The present invention clearly extends to the use of multiple structural gene regions which preferably comprise a direct repeat sequence, inverted repeat sequence or interrupted palindrome sequence of a particular structural gene, dispersed nucleic acid molecule or foreign nucleic acid molecule, or a fragment thereof. - 30 -
Each dispersed or foreign nucleic acid molecule contained within the multiple structural gene unit of the subject synthetic gene may comprise a nucleotide sequence which is substantially identical to a different target gene in the same organism. Such an arrangement may be of particular utility when the synthetic gene is intended to provide protection against a pathogen in a cell, tissue or organ, in particular a viral pathogen, by modifying the expression of viral target genes. For example, the multiple structural gene may comprise nucleotide sequences (i.e. two or more dispersed or foreign nucleic acid molecules) which are substantially identical to two or more target genes selected from the list comprising DNA polymerase, RNA polymerase, Nia protease, and coat protein or other target gene which is essential for viral infectivity, replication or reproduction. However, it is preferred with this arrangement that the structural gene units are selected such that the target genes to which they are substantially identical are normally expressed at approximately the same time (or later) in an infected cell, tissue or organ as (than) the multiple structural gene of the subject synthetic gene is expressed under control of the promoter sequence. This means that the promoter controlling expression of the multiple structural gene will usually be selected to confer expression in the cell, tissue or organ over the entire life cycle of the virus when the viral target genes are expressed at different stages of infection.
As with the individual sequence units of a dispersed or foreign nucleic acid molecule, the individual units of the multiple structural gene may be spatially connected in any orientation relative to each other, for example head-to-head, head-to-tail or tail-to-tail and all such configurations are within the scope of the invention.
For expression in eukaryotic cells, the synthetic gene generally comprises, in addition to the nucleic acid molecule of the invention, a promoter and optionally other regulatory sequences designed to facilitate expression of the dispersed nucleic acid molecule or foreign nucleic acid molecule.
Reference herein to a "promoter" is to be taken in its broadest context and includes the 31
transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene region, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell.
Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the sense molecule and/or to alter the spatial expression and/or temporal expression of said sense molecule. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense molecule, thereby conferring copper inducibility on the expression of said molecule.
Placing a dispersed or foreign nucleic acid molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous - 32
gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
Examples of promoters suitable for use in the synthetic genes of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in plant, animal, insect, fungal, yeast or bacterial cells. The promoter may regulate the expression of the structural gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or pathogens, or metal ions, amongst others.
Preferably, the promoter is capable of regulating expression of a nucleic acid molecule in a eukaryotic cell, tissue or organ, at least during the period of time over which the target gene is expressed therein and more preferably also immediately preceding the commencement of detectable expression of the target gene in said cell, tissue or organ.
Accordingly, strong constitutive promoters are particularly preferred for the purposes of the present invention or promoters which may be induced by virus infection or the commencement of target gene expression.
Plant-operable and animal-operable promoters are particularly preferred for use in the synthetic genes of the present invention. Examples of preferred promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator- promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, CaMV 35S promoter, SCSV promoter, SCBV promoter and the like.
In consideration of the preferred requirement for high-level expression which coincides with expression of the target gene or precedes expression of the target gene, it is - 33
highly desirable that the promoter sequence is a constitutive strong promoter such as the CMV-IE promoter or the SV40 early promoter sequence, the SV40 late promoter sequence, the CaMV 35S promoter, or the SCBV promoter, amongst others. Those skilled in the art will readily be aware of additional promoter sequences other than those specifically described.
In the present context, the terms "in operable connection with" or "operably under the control" or similar shall be taken to indicate that expression of the structural gene region or multiple structural gene region is under the control of the promoter sequence with which it is spatially connected; in a cell, tissue, organ or whole organism.
In a preferred embodiment of the invention, a structural gene region (i.e. dispersed nucleic acid molecule or foreign nucleic acid molecule) or multiple structural gene region is placed operably in connection with a promoter orientation relative to the promoter sequence, such that when it is transcribed an mRNA product is synthesized which, if translated, is capable of encoding a polypeptide product of the target gene or a fragment thereof.
However, the present invention is not to be limited to the use of such an arrangement and the invention clearly extends to the use of synthetic genes and genetic constructs wherein the a structural gene region or multiple structural gene region is placed in the "antisense" orientation relative to the promoter sequence, such that at least a part of the mRNA transcription product thereof is complementary to the mRNA encoded by the target gene or a fragment thereof.
Clearly, as the dispersed nucleic acid molecule, foreign nucleic acid molecule or multiple structural gene region comprises tandem direct and/or inverted repeat sequences of the target gene, all combinations of the above-mentioned configurations are encompassed by the invention. 34
In an alternative embodiment of the invention, the structural gene region or multiple structural gene region is operably connected to both a first promoter sequence and a second promoter sequence, wherein said promoters are located at the distal and proximal ends thereof such that at least one unit of said a structural gene region or multiple structural gene region is placed in the "sense" orientation relative to the first promoter sequence and in the "antisense" orientation relative to the second promoter sequence. According to this embodiment, it is also preferred that the first and second promoters be different, to prevent competition there between for cellular transcription factors which bind thereto. The advantage of this arrangement is that the effects of transcription from the first and second promoters in reducing target gene expression in the cell may be compared to determine the optimum orientation for each nucleotide sequence tested.
The synthetic gene preferably contains additional regulatory elements for efficient transcription, for example a transcription termination sequence.
The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants or synthesized de novo.
As with promoter sequences, the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used.
Examples of terminators particularly suitable for use in the synthetic genes of the present invention include the SV40 polyadenylation signal, the HSV TK polyadenylation signal, the CYC1 terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) gene terminator of Agrobactehum tumefaciens, the - 35 -
terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit gene (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any t ?o-independent E.coli terminator, or the lacZ alpha terminator, amongst others.
In a particularly preferred embodiment, the terminator is the SV40 polyadenylation signal or the HSVTK polyadenylation signal which are operable in animal cells, tissues and organs, octopine synthase (OCS) or nopaline synthase (NOS) terminator active in plant cells, tissues or organs, or the lacZ alpha terminator which is active in prokaryotic cells.
Those skilled in the art will be aware of additional terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.
Means for introducing (i.e. transfecting or transforming) cells with the synthetic genes described herein or a genetic construct comprising same are well-known to those skilled in the art.
In a further alternative embodiment, a genetic construct is used which comprises two or more structural gene regions or multiple structural gene regions wherein each of said structural gene regions is placed operably under the control of its own promoter sequence. As with other embodiments described herein, the orientation of each structural gene region may be varied to maximise its modulatory effect on target gene expression.
According to this embodiment, the promoters controlling expression of the structural gene unit are preferably different promoter sequences, to reduce competition there between for cellular transcription factors and RNA polymerases. Preferred promoters are selected from those referred to supra. - 36 -
Those skilled in the art will know how to modify the arrangement or configuration of the individual structural genes as described supra to regulate their expression from separate promoter sequences.
The synthetic genes described supra are capable of being modified further, for example by the inclusion of marker nucleotide sequences encoding a detectable marker enzyme or a functional analogue or derivative thereof, to facilitate detection of the synthetic gene in a cell, tissue or organ in which it is expressed. According to this embodiment, the marker nucleotide sequences will be present in a translatable format and expressed, for example as a fusion polypeptide with the translation product(s) of any one or more of the structural genes or alternatively as a non-fusion polypeptide.
Those skilled in the art will be aware of how to produce the synthetic genes described herein and of the requirements for obtaining the expression thereof, when so desired, in a specific cell or cell-type under the conditions desired. In particular, it will be known to those skilled in the art that the genetic manipulations required to perform the present invention may require the propagation of a genetic construct described herein or a derivative thereof in a prokaryotic cell such as an E. coli cell or a plant cell or an animal cell.
The synthetic genes of the present invention may be introduced to a suitable cell, tissue or organ without modification as linear DNA in the form of a genetic construct, optionally contained within a suitable carrier, such as a cell, virus particle or liposome, amongst others. To produce a genetic construct, the synthetic gene of the invention is inserted into a suitable vector or episome molecule, such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced.
Accordingly a further aspect of the invention provides a genetic construct which at 37 -
least comprises the synthetic gene according to any one or more of the embodiments described herein and one or more origins of replication and/or selectable marker gene sequences.
Genetic constructs are particularly suitable for the transformation of a eukaryotic cell to introduce novel genetic traits thereto, in addition to the provision of resistance characteristics to viral pathogens. Such additional novel traits may be introduced in a separate genetic construct or, alternatively on the same genetic construct which comprises the synthetic genes described herein. Those skilled in the art will recognise the significant advantages, in particular in terms of reduced genetic manipulations and tissue culture requirements and increased cost-effectiveness, of including genetic sequences which encode such additional traits and the synthetic genes described herein in a single genetic construct.
Usually, an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to a eukaryotic cell, or integrated into the genome of a eukaryotic cell.
In a particularly preferred embodiment, the origin of replication is functional in a bacterial cell and comprises the pUC or the ColE1 origin or alternatively the origin of replication is operable in a eukaryotic cell, tissue and more preferably comprises the 2 micron (2μxn) origin of replication or the SV40 origin of replication.
As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.
Suitable selectable marker genes contemplated herein include the ampiciliin-resistance 38
gene (Amp"), tetracycline-resistance gene (Tcr), bacterial kanamycin-resistance gene (Kanr), is the zeocin resistance gene (Zeocin is a drug of bleomycin family which is trademark of InVitrogen Corporation), the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gene (nptW), hygromycin-resistance gene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein- encoding gene or the luciferase gene, amongst others.
Preferably, the selectable marker gene is the npfll gene or Kanr gene or green fluorescent protein (GFP)-encoding gene.
Those skilled in the art will be aware of other selectable marker genes useful in the performance of the present invention and the subject invention is not limited by the nature of the selectable marker gene.
The present invention extends to all genetic constructs essentially as described herein, which include further genetic sequences intended for the maintenance and/or replication of said genetic construct in prokaryotes or eukaryotes and/or the integration of said genetic construct or a part thereof into the genome of a eukaryotic cell or organism.
As with dispersed or foreign nucleic acid molecules, standard methods described supra may be used to introduce synthetic genes and genetic constructs into the cell, tissue or organ for the purposes of modulating the expression of the target gene, for example liposome-mediated transfection or transformation, transformation of cells with attenuated virus particles or bacterial cells, cell mating, transformation or transfection procedures known to those skilled in the art or described by Ausubel et al. (1992).
Additional means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCI2 and variations thereof, in particular - 39 -
the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski er a/, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990) microparticle bombardment, electroporation (Fromm et al., 1985), microinjection of DNA (Crossway er a/., 1986), microparticle bombardment of tissue explants or cells (Christou etal, 1988; Sanford, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobactehum to the plant tissue as described essentially by An et a/.(1985), Herrera-Estrella et al. (1983a, 1983b, 1985).
For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp etal. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
In a further embodiment of the present invention, the synthetic genes and genetic constructs described herein are adapted for integration into the genome of a cell in which it is expressed. Those skilled in the art will be aware that, in order to achieve integration of a genetic sequence or genetic construct into the genome of a host cell, certain additional genetic sequences may be required. In the case of plants, left and right border sequences from the T-DNA of the Agrobactehum tumefaciens Ti plasmid will generally be required.
The present invention further extends to an isolated cell, tissue or organ comprising 40
the synthetic gene described herein or a genetic construct comprising same. The present invention extends further to regenerated tissues, organs and whole organisms derived from said cells, tissues and organs and to propagules and progeny thereof.
For example, plants may be regenerated from transformed plant cells or tissues or organs on hormone-containing media and the regenerated plants may take a variety of forms, such as chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). Transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.
The present invention is further described with reference to the following non-limiting Examples.
- 41
EXAMPLE 1
Genetic constructs comprising BEV polymerase gene sequences linked to the CMV promoter sequence and/or the SV40L promoter sequence
1. Commercial Plasmids Plasmid pBluescript II (SK+)
Plasmid pBluescript II (SK+) is commercially available from Stratagene and comprises the LacZ promoter sequence and /acZ-alpha transcription terminator, with a multiple cloning site for the insertion of structural gene sequences therein. The plasmid further comprises the ColE1 and fl origins of replication and ampiciliin-resistance gene.
Plasmid pSVL
Plasmid pSVL is commercially-obtainable from Pharmacia and serves as a source of the SV40 late promoter sequence. The nucleotide sequence of pSVL is also publicly available as GenBank Accession Number U13868.
Plasmid pCR2.1
Plasmid pCR2.1 is commercially available from Invitrogen and comprises the LacZ promoter sequence and lacZ-α transcription terminator, with a cloning site for the insertion of structural gene sequences there between. Plasmid pCR2.1 is designed to clone nucleic acid fragments by virtue of the A-overhang frequently synthesized by Taq polymerase during the polymerase chain reaction. PCR fragments cloned in this fashion are flanked by two EcoRI sites. The plasmid further comprises the ColE1 and fl origins of replication and kanamycin-resistance and ampiciliin-resistance genes.
Plasmid pEGFP-N1 MCS
Plasmid pEGFP-N1 MCS (Figure 1; Clontech) contains the CMV IE promoter operably connected to an open reading frame encoding a red-shifted variant of wild-type green fluorescent protein (GFP; Prasher et al., 1992; Chalfie et al., 1994; Inouye and Tsuji, - 42 -
1994), which has been optimised for brighter fluorescence. The specific GFP variant encoded by pEGFP-N1 MCS has been disclosed by Cormack et al. (1996). Plasmid pEGFP-N1 MCS contains a multiple cloning site comprising BglW and Bam \ sites and many other restriction endonuclease cleavage sites, located between the CMV IE promoter and the GFP open reading frame. Structural genes cloned into the multiple cloning site will be expressed at the transcriptional level if they lack a functional translation start site, however such structural gene sequences will not be expressed at the protein level (i.e. translated). Structural gene sequences inserted into the multiple cloning site which comprise a functional translation start site will be expressed as GFP fusion polypeptides if they are cloned in-frame with the GFP-encoding sequence. The plasmid further comprises an SV40 polyadenylation signal downstream of the GFP open reading frame to direct proper processing of the 3'-end of mRNA transcribed from the CMV-IE promoter sequence. The plasmid further comprises the SV40 origin of replication functional in animal cells; the neomycin-resistance gene comprising SV40 early promoter (SV40 EP in Figure 1) operably connected to the neomycin/kanamycin-resistance gene derived from Tn5 (Kan/neo in Figure 1) and the HSV thymidine kinase polyadenylation signal (HSV TK poly (A) in Figure 1), for selection of transformed cells on kamanycin, neomycin or G418; the pUC19 origin of replication which is functional in bacterial cells (pUC Ori in Figure 1); and the f1 origin of replication for single-stranded DNA production (fl Ori in Figure 1).
2. Expression cassettes Plasmid pCMV.cass
Plasmid pCMV.cass (Figure 2) is an expression cassette for driving expression of a structural gene sequence under control of the CMV-IE promoter sequence. Plasmid pCMV.cass was derived from pEGFP-N1 MCS by deletion of the GFP open reading frame as follows: Plasmid pEGFP-N1 MCS was digested with PinA and Not I, blunt- ended using Pfu\ polymerase and then re-ligated. Structural gene sequences are cloned into pCMV.cass using the multiple cloning site, which is identical to the multiple cloning site of pEGFP-N1 MCS, except it lacks the P/'nAI site. 43
Plasmid pCMV.SV40L.cass
Plasmid pCMV.SV40L.cass (Figure 3) comprises the synthetic poly A site and the SV40 late promoter sequence from plasmid pCR.SV40L (Figure 4), sub-cloned as a Sal I fragment, into the Sal I site of plasmid pCMV.cass (Figure 2), such that the CMV- IE promoter and SV40 late promoter sequences are capable of directing transcription in the same direction. Accordingly, the synthetic poly(A) site at the 5' end of the SV40 promoter sequence is used as a transcription terminator for structural genes expressed from the CMV IE promoter in this plasmid, which also provides for the insertion of said structural gene via the multiple cloning site present between the SV40 late promoter and the synthetic poly(A) site (Figure 5). The multiple cloning sites are located behind the CMV-IE and SV40 late promoters, including Bam λ\ and BglW sites.
Plasmid pCMV.SV40LR.cass
Plasmid pCMV.SV40LR.cass (Figure 4) comprises the SV40 late promoter sequence derived from plasmid pCR.SV40L, sub-cloned as a Sail fragment into the Sa/I site of the plasmid pCMV.cass (Figure 2), such that the CMV-IE or the SV40 late promoter may drive transcription of a structural gene or a multiple structural gene unit, in the sense or antisense orientation, as desired. A multiple cloning site is positioned between the opposing CMV- IE and SV40 late promoter sequences in this plasmid to facilitate the introduction of a structural gene sequence. In order for expression of a structural gene sequence to occur from this plasmid, it must be introduced with its own transcription termination sequence located at the 3' end, because there are no transcription termination sequences located between the opposing CMV- IE and SV40 late promoter sequences in this plasmid. Preferably, the structural gene sequence or multiple structural gene unit which is to be introduced into pCMV.SV40LR.cass will comprise both a 5' and a 3' polyadenylation signal as follows:
(i) where the structural gene sequence or multiple structural gene unit is to be expressed in the sense orientation from the CMV IE promoter sequence and/or in the antisense orientation from the SV40 late promoter, the 5' polyadenylation signal will be in the antisense orientation and the 3' 44
polyadenylation signal will be in the sense orientation; and (ii) where the structural gene sequence or multiple structural gene unit is to be expressed in the antisense orientation from the CMV IE promoter sequence and/or in the sense orientation from the SV40 late promoter, the 5' polyadenylation signal will be in the sense orientation and the 3' polyadenylation signal will be in the antisense orientation.
Alternatively or in addition, suitably-oriented terminator sequences may be placed at the 5'-end of the CMV and SV40L promoters, as shown in Figure 4.
Alternatively, plasmid pCMV.SV40LR.cass is further modified to produce a derivative plasmid which comprises two polyadenylation signals located between the CMV IE and SV40 late promoter sequences, in approriate orientations to facilitate expression of any structural gene located therebetween in the sense or antisense orientation from either the CMV IE promoter or the SV40 promoter sequence. The present invention clearly encompasses such derivatives.
Alternatively appropriately oriented terminators could be placed upstream of the CMV and SV40L promoters such that transcriptional termination could occur after readthrough of each of the two promoters in the antisense orientation.
3. Intermediate Constructs Plasmid pCR.Bgl-GFP-Bam
Plasmid pCR.Bgl-GFP-Bam (Figure 5) comprises an internal region of the GFP open reading frame derived from plasmid pEGFP-N1 MCS (Figure 1) placed operably under the control of the lacZ promoter. To produce this plasmid, a region of the GFP open reading frame was amplified from pEGFP-N1 MCS using the amplification primers Bgl- GFP and GFP-Bam and cloned into plasmid pCR2.1. The internal GFP-encoding region in plasmid pCR.Bgl-GFP-Bam lacks functional translational start and stop codons. - 45 -
Plasmid pBSII(SK+).EGFP
Plasmid pBSII(SK+).EGFP (Figure 6) comprises the EGFP open reading frame derived from plasmid pEGFP-N1 MCS (Figure 1) placed operably under the control of the lacZ promoter. To produce this plasmid, the EGFP encoding region of pEGFP-N1 MCS was excised as a NotMXhoλ fragment and cloned into the NotMXho cloning sites of plasmid pBluescript II (SK+).
Plasmid pCMV.EGFP
Plasmid pCMV.EGFP (Figure 7) is capable of expressing the EGFP structural gene under the control of the CMV-IE promoter sequence. To produce this plasmid the EGFP sequence from plasmid pBSII(SK+).EGFP was excised as BamHUSacl fragment and cloned into the BglU/Sacl sites of plasmid pCMV.cass (Figure 2).
Plasmid pCR.SV40L Plasmid pCR.SV40L (Figure 8) comprises the SV40 late promoter derived from plasmid pSVL (GenBank Accession No. U 13868; Pharmacia), cloned into pCR2.1 (Stratagene). To produce this plasmid, the SV40 late promoter was amplified using the primers SV40-1 and SV40-2 which comprise Sal I cloning sites to facilitate sub- cloning of the amplified DNA fragment into pCMV.cass. The primer also contains a synthetic poly (A) site at the 5' end, such that the amplicification product comprises the synthetic poly(A) site at the 5' end of the SV40 promoter sequence.
Plasmid pCR.BEV.1
The BEV RNA-dependent RNA polymerase coding region was amplified as a 1 ,385 bp DNA fragment from a full-length cDNA clone encoding same, using primers designated BEV-1 and BEV-2, under standard amplification conditions. The amplified DNA contained a 5'-Bgl II restriction enzyme site, derived from the BEV-1 primer sequence and a 3'βamHI restriction enzyme site, derived from the BEV-2 primer sequence. Additionally, as the BEV-1 primer sequence contains a translation start signal 5'-ATG-3' engineered at positions 15-17, the amplified BEV polymerase 46 -
structural gene comprises the start site in-frame with BEV polymerase-encoding nucleotide sequences, Thus, the amplified BEV polymerase structural gene comprises the ATG start codon immediately upstream (ie. juxtaposed) to the BEV polymerase- encoding sequence. There is no translation stop codon in the amplified DNA. This plasmid is present as Figure 9.
Plasmid pCR.BEV.2
The complete BEV polymerase coding region was amplified from a full-length cDNA clone encoding same, using primers BEV-1 and BEV-3. Primer BEV-3 comprises a BamHl restriction enzyme site at positions 5 to 10 inclusive and the complement of a translation stop signal at positions 11 to 13. As a consequence, an open reading frame comprising a translation start signal and translation stop signal, contained between the Bgl II and BamHl restriction sites. The amplified fragment was cloned into pCR2J (Stratagene) to produce plasmid pCR2.BEV.2 (Figure 10).
Plasmid pCR.BEV.3
A non-translatable BEV polymerase structural gene was amplified from a full-length BEV polymerase cDNA clone using the amplification primers BEV-3 and BEV-4. Primer BEV-4 comprises a Bglll cloning site at positions 5-10 and sequences downstream of this Bglll site are homologous to nucleotide sequences of the BEV polymerase gene. There is no functional ATG start codon in the amplified DNA product of primers BEV-3 and BEV-4. The BEV polymerase is expressed as part of a polyprotein and, as a consequence, there is no ATG translation start site in this gene. The amplified DNA was cloned into plasmid pCR2.1 (Stratagene) to yield plasmid pCR.BEV.3 (Figure 11).
Plasmid pCMV.EGFP.BEV2
Plasmid pCMV.EGFP.BEV2 (Figure 12) was produced by cloning the BEV polymerase sequence from pCR.BEV.2 as a Bglll/BamHI fragment into the BamHl site of pCMV.EGFP. 47
4. Control Plasmids Plasmid pCMV.BEV.2
Plasmid pCMV.BEV.2 (Figure 13) is capable of expressing the entire BEV polymerase open reading frame under the control of CMV-IE promoter sequence. To produce pCMV.BEV.2, the BEV polymerase sequence from pCR.BEV.2 was sub-cloned in the sense orientation as a Bg/ll-to-SamHI fragment into Sg/ll/SamHI-digested pCMV.cass (Figure 2).
Plasmid pCMV.BEV.3 Plasmid pCMV.BEV.3 (Figure 14) expresses a non-translatable BEV polymerase structural gene in the sense orientation under the control of the CMV-IE promoter sequence. To produce pCMV.BEVnt, the BEV polymerase sequence from pCR.BEV.3 was sub-cloned in the sense orientation as a Sg/ll-to-SamHI fragment into Bg/ll/βamHI-digested pCMV.cass (Figure 2).
Plasmid pCMV.VEB
Plasmid pCMV.VEB (Figure 15) expresses an antisense BEV polymerase mRNA under the control of the CMV-IE promoter sequence. To produce plasmid pCMV.VEB, the BEV polymerase sequence from pCR.BEV.2 was sub-cloned in the antisense orientation as a Sg/ll-to-SamHI fragment into βg/ll/SamHI-digested pCMV.cass (Figure 2).
Plasmid pCMV.BEV.GFP
Plasmid pCMV.BEV.GFP (Figure 16) was constructed by cloning the GFP fragment from pCR.Bgl-GFP-Bam as a Bglll/BamHI fragment into BamHI-digested pCMV.BEV.2. This plasmid serves as a control in some experiments and also as an intermediate construct.
Plasmid pCMV.BEV.SV40-L Plasmid pCMV.BEV.SV40-L (Figure 17) comprises a translatable BEV polymerase 48 -
structural gene derived from plasmid pCR.BEV.2 inserted in the sense orientation between the CMV-IE promoter and the SV40 late promoter sequences of plasmid pCMV.SV40L.cass. To produce plasmid pCMV.BEV.SV40L-O, the BEV polymerase structural gene was sub-cloned as a Bg/ll-to-BamHI fragment into Bg/ll-digested pCMV.SV40L.cass DNA.
Plasmid pCMV.O.SV40L.BEV
Plasmid pCMV.O.SV40L.BEV (Figure 18) comprises a translatable BEV polymerase structural gene derived from plasmid pCR.BEV.2 cloned downstream of tandem CMV- IE promoter and SV40 late promoter sequences present in plasmid pCMV.SV40L.cass. To produce plasmid pCMV.O.SV40L.BEV, the BEV polymerase structural gene was sub-cloned in the sense orientation as a Bg/ll-to-BamHI fragment into BamHI-digested pCMV.SV40L.cass DNA.
Plasmid pCMV.O.SV40L.VEB
Plasmid pCMV.O.SV40L.VEB (Figure 19) comprises an antisense BEV polymerase structural gene derived from plasmid pCR.BEV.2 cloned downstream of tandem CMV- IE promoter and SV40 late promoter sequences present in plasmid pCMV.SV40L.cass. To produce plasmid pCMV.O.SV40LVEB, the BEV polymerase structural gene was sub-cloned in the antisense orientation as a Bg/ll-to-BamHI fragment into BamHI-digested pCMV.SV40L.cass DNA.
5. Test Plasmids Plasmid pCMV.BEVx2 Plasmid pCMV.BEVx2 (Figure 20) comprises a direct repeat of a complete BEV polymerase open reading frame under the control of the CMV-IE promoter sequence. In eukaryotic cells at least, the open reading frame located nearer the CMV-IE promoter is translatable. To produce pCMV.BEVx2, the BEV polymerase structural gene from plasmid pCR.BEV.2 was sub-cloned in the sense orientation as a Bg/ll-to- BamHl fragment into BamHI-digested pCMV.BEV.2, immediately downstream of the 49 -
BEV polymerase structural gene already present therein.
Plasmid pCMV.BEVx3
Plasmid pCMV.BEVx3 (Figure 21) comprises a direct repeat of three complete BEV polymerase open reading frames under the control of the CMV-1E promoter. To produce pCMV.BEVx3, the BEV polymerase fragment from pCR.BEV.2 was cloned in the sense orientation as a Bglll/BamHI fragment into the BamHl site of pCMV.BEVx2, immediately downstream of the BEV polymerase sequences already present therein.
Plasmid pCMV.BEVx4
Plasmid pCMV.BEVx4 (Figure 22) comprises a direct repeat of four complete BEV polymerase open reading frames under the control of the CMV-1 E promoter. To produce pCMV.BEVx4, the BEV polymerase fragment from pCR.BEV.2 was cloned in the sense orientation as a Bglll/BamHI fragment into the BamHl site of pCMV.BEVx3, immediately downstream of the BEV polymerase sequences already present therein.
Plasmid pCMV.BEV.SV40L.BEV Plasmid pCMV.BEV.SV40L.BEV(Figure 23) comprises a multiple structural gene unit comprising two BEV polymerase structural genes placed operably and separately under control of the CMV-IE promoter and SV40 late promoter sequences. To produce plasmid pCMV.BEV.SV40L.BEV, the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned in the sense orientation as a Bg/ll-to- BamHl fragment behind the SV40 late promoter sequence present in BamHI-digested pCMV.BEV.SV40L-O.
Plasmid pCMV.BEV.SV40L.VEB
Plasmid pCMV.BEV.SV40L.VEB (Figure 24) comprises a multiple structural gene unit comprising two BEV polymerase structural genes placed operably and separately - 50 -
under control of the CMV-IE promoter and SV40 late promoter sequences. To produce plasmid pCMV.BEV.SV40L.VEB, the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned in the antisense orientation as a Bg/ll-to- BamHI fragment behind the SV40 late promoter sequence present in BamHI-digested pCMV.BEV.SV40L-O. In this plasmid, the BEV polymerase structural gene is expressed in the sense orientation under control of the CMV-IE promoter to produce a translatable mRNA, whilst the BEV polymerase structural gene is also expressed under control of the SV40 promoter to produce an antisense mRNA species.
Plasmid pCMV.BEV.GFP.VEB
Plasmid pCMV.BEV.GFP.VEB (Figure 25) comprises a BEV structural gene inverted repeat or palindrome, interrupted by the insertion of a GFP open reading frame (stuffer fragment) between each BEV structural gene sequence in the inverted repeat. To produce plasmid pCMV.BEV.GFP.VEB, the GFP stuffer fragment from pCR.Bgl-GFP- Bam was first sub-cloned in the sense orientation as a Bg/ll-to-BamHI fragment into BamHI-digested pCMV.BEV.2 to produce an intermediate plasmid pCMV.BEV.GFP wherein the BEV polymerase-encoding and GFP-encoding sequences are contained within the same 5'-Bg/ll-to-BamHI-3' fragment. The BEV polymerase structural gene from pCMV.BEV.2 was then cloned in the antisense orientation as a Bg/ll-to-BamHI fragment into BamHI-digested pCMV.BEV.GFP. The BEV polymerase structural gene nearer the CMV-IE promoter sequence in plasmid pCMV.BEV.GFP.VEB is capable of being translated, at least in eukaryotic cells.
Plasmid pCMV.EGFP.BEV2.PFG Plasmid pCMV.EGFP.BEV2.PFG (Figure 26) comprise a GFP palindrome, interrupted by the insertion of a BEV polymerase sequence between each GFP structural gene in the inverted repeat. To produce this plasmid the GFP fragment from pCR.Bgl-GFP- Bam was cloned as a Bglll/BamHI fragment into the BamHl site of pCMV.EGFP.BEV2 in the antisense orientation relative to the CMV promoter. - 51 -
Plasmid pCMV.BEV.SV40LR
Plasmid pCMV.BEV.SV40LR (Figure 27) comprises a structural gene comprising the entire BEV polymerase open reading frame placed operably and separately under control of opposing CMV-IE promoter and SV40 late promoter sequences, thereby potentially producing BEV polymerase transcripts at least from both strands of the full- length BEV polymerase structural gene. To produce plasmid pCMV.BEV.SV40LR, the translatable BEV polymerase structural gene present in pCR.BEV.2 was sub-cloned, as a Bg/ll-to-BamHI fragment, into the unique Bglll site of plasmid pCMV.SV40LR.cass, such that the BEV open reading frame is present in the sense orientation relative to the CMV-IE promoter sequence.
Those skilled in the art will recognise that it is possible to generate a plasmid wherein the BEV polymerase fragment from pCR.BEV.2 is inserted in the antisense orientation, relative to the CMV IE promoter sequence, using this cloning strategy. The present invention further encompasses such a genetic construct.
EXAMPLE 2 Genetic constructs comprising the porcine α-1,3-galactosyltransferase (Gait) structural gene sequence or sequences operably connected to the CMV promoter sequence and/or the SV40L promoter sequence
1. Commercial Plasmids Plasmid pcDNA3
Plasmid pcDNA3 is commercially available from Invitrogen and comprises the CMV-IE promoter and BGHpA transcription terminator, with multiple cloning sites for the insertion of structural gene sequences there between. The plasmid further comprises the ColE1 and fl origins of replication and neomycin-resistance and ampiciliin- resistance genes. 52
2. Intermediate plasmids Plasmid pcDNA3.Galt
Plasmid pcDNA3.Galt (BresaGen Limited, South Australia, Australia; Figure 28) is plasmid pcDNA3 (Invitrogen) and comprises the cDNA sequence encoding porcine gene alpha-1,3-galactosyltransferase (Gait) operably under the control of the CMV-IE promoter sequence such that it is capable of being expressed therefrom. To produce plasmid pcDNA3.Galt, the porcine gene alpha-1 ,3-galactosyltransferase cDNA was cloned as an EcoRI fragment into the EcoRI cloning site of pcDNA3. The plasmid further comprises the ColE1 and fl origins of replication and the neomycin and ampiciliin-resistance genes.
3. Control Plasmids Plasmid pCMV.Galt
Plasmid pCMV.Galt (Figure 29) is capable of expressing the Gait structural gene under the control of the CMV-IE promoter sequence. To produce plasmid pCMV.Galt, the
Gait sequence from plasmid pcDNA3.Galt was excised as an EcoRI fragment and cloned in the sense orientation into the EcoRI site of plasmid pCMV.cass (Figure 2).
Plasmid pCMV.EGFP.Galt Plasmid pCMV.EGFP.Galt (Figure 30) is capable of expressing the Gait structural gene as a Gait fusion polypeptide under the control of the CMV-IE promoter sequence. To produce plasmid pCMV.EGFP.Galt, the Gait sequence from pCMV.Galt (Figure 29) was excised as a Bglll/BamHI fragment and cloned into the BamHl site of pCMV.EGFP.
Plasmid pCMV.Galt.GFP
Plasmid pCMV.Galt.GFP (Figure 31) was produced by cloning the Gait cDNA as an EcORI fragment from pCDNA3 into EcoRI-digested pCMV.EGFP in the sense orientation. This plasmid serves as both a control and construct intermediate. 53
Plasmid pCMV.Galt.SV40L.O
The plasmid pCMV.Galt.SV40L.0 (Figure 32) comprises a Gait structural gene cloned downstream of the CMV promoter present in pCMV.SV40L.cass. To produce the plasmid the Gait cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHI into Bglll-digested pCMV.SV40L.cass in the sense orientation.
Plasmid pCMV.O.SV40L.tlaG
The plasmid pCMV.O.SV40L.tlaG (Figure 33) comprises a Gait structural gene clones in an antisense orientation downstream of the SV40L promoter present in pCMV.SV40L.cass. To produce this plasmid the Gait cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHI into BamHI-digested pCMV.SV40L.cass in the antisense orientation.
Plasmid pCMV.O.SV40L.Galt The plasmid pCMV.O.SV40L.Galt (Figure 34) comprises a Gait structural gene cloned downstream of the SV40L promoter present in pCMV.SV40L.cass. To produce the plasmid the Gait cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHI into BamHI-digested pCMV.SV40L.cass in the sense orientation.
4. Test Plasmids Plasmid pCMV.Galtx2
Plasmid pCMV.Galtx2 (Figure 35) comprises a direct repeat of a Gait open reading frame under the control of the CMV-IE promoter sequence. In eukaryotes cells at least, the open reading frame located nearer the CMV-IE promoter is translatable. To produce pCMV.Galtx2, the Gait structural gene from pCMV.Galt was excised as a Bglll/BamHI fragment and cloned in the sense orientation into the BamHl cloning site of pCMV.Galt.
Plasmid pCMV.Galtx4 Plasmid pCMV.Galtx4 (Figure 36) comprises a quadruple direct repeat of a Gait open - 54 -
reading frame under the control of the CMV-IE promoter sequence. In eukaryotes cells at least, the open reading frame located nearer the CMV-IE promoter is translatable. To produce pCMV.Galtx4, the Galtx2 sequence from pCMV.Galtx2 was excised as a Bglll/BamHI fragment and cloned in the sense orientation into the BamHl cloning site of pCMV.Galtx2.
Plasmid pCMV.Galt.SV40L.Galt
The plasmid pCMV.Galt.SV40L.Galt (Figure 37) is designed to express two sense transcripts of Gait, one driven by the CMV promoter, the other by the SV40L promoter. To produce the plasmid a Gait cDNA fragment from pCMV.Galt was cloned as a
Bglll/BamHI fragment into Bg Ill-digested pCMV.O.SV40.Galt in the sense orientation.
Plasmid pCMV.Galt.SV40L.tlaG
The plasmid pCMV.Galt.SV40.tlaG (Figure 38) is designed to express a sense transcript of Gait driven by the CMV promoter and an antisense transcript driven by the SV40L promoter. To produce the plasmid a Gait cDNA fragment from pCMV.Galt was cloned as a Bglll/BamHI fragment into Bglll-digested pCMV.O.SV40.talG in the sense orientation.
Plasmid pCMV.Galt.GFP.tlaG
Plasmid pCMV.Galt.GFP.tlaG (Figure 39) comprise a Gait palindrome, interrupted by the insertion of a GFP sequence between each Gait structural gene in the inverted repeat. To produce this plasmid the Bglll/BamHI Gait cDNA fragment from pCMV.Galt was cloned into the BamHl site of pCMV.Galt.GFP in the antisense relative to the CMV promoter.
Plasmid pCMV.EGFP.Galt.PFG
The plasmid pCMV.EGFP.Galt.PFG (Figure 40) comprises a GFP palindrome, interrupted by the insertion of a Gait sequence between each GFP structural gene of the inverted repeat, expression of which is driven by the CMV promoter. To produce 55
this plasmid the Gait sequences from pCMV.Galt were cloned as a Bglll/BamHI fragment into BamHI-digested pCMV.EGFP in the sense orientation to produce the intermediate pCMV.EGFP.Galt (not shown); following this further GFP sequences from pCR.Bgl-pCMV.EGFP.Galt in the antisense orientation.
Plasmid pCMV.Galt.SV40LR
The plasmid pCMV.Galt.SV40LR (Figure 41) is designed to express GalT cDNA sequences cloned between the opposing CMV and SV40L promoters in the expression cassette pCMV.SV40LR.cass. To produce this plasmid Gait sequences from pCMV.Galt were cloned as a Bglll/BamHI fragment in Bg Ill-digested pCMV.SV40LR.cass in the sense orientation relative to the 35S promoter.
EXAMPLE 3 Genetic constructs comprising PVY Nia sequences operably linked to the35S promoter sequence and/or the SCBV promoter sequence
1: Binary vector Plasmid pART27
Plasmid pART27 is a binary vector, specifically designed to be compatible with the pART7 expression cassette. It contains bacterial origins of replication for both E. coli and Agrobacterium tumefaciens, a spectinomycin resistance gene for bacterial selection, left and right T-DNA borders for transfer of DNA from Agrobacterium to plant cells and a kanamycin resistance cassette to permit selection of transformed plant cells. The kanamycin resistance cassette is located between the T-DNA borders, pART27 also contains a unique NotI restriction site which permits cloning of constructs prepared in vectors such as pART7 to be cloned between the T-DNA borders. Construction of pART27 is described in Gleave, AP (1992).
When cloning NotI inserts into this vector, two insert orientations can be obtained. In all the following examples the same insert orientation, relative to the direction of the - 56 -
35S promoter in the described pART7 constructs was chosen; this was done to minimise any experimental artefacts that may arise from comparing different constructs with different insert orientations.
2. Commercial plasmids Plasmid pBC (KS-)
Plasmid pBC (KS-) is commercially available from Stratagene and comprises the LacZ promoter sequence and lacZ-alpha transcription terminator, with a multiple cloning site for the insertion of structural gene sequences therein. The plasmid further comprises the ColE1 and fl origins of replication and a chloroamphenicol-resistance gene.
Plasmid pSP72
Plasmid pSP72 is commercially available from Promega and contains a multiple cloning site for the insertion of structural gene sequences therein. The plasmid further comprises the ColE1 origin of replication and an ampiciliin-resistance gene.
3. Expression cassettes Plasmid pART7
Plasmid pART7 is an expression cassette designed to drive expression of sequences cloned behind the 35S promoter. It contains a polylinkerto assist cloning and a region of the octipine synthase terminator. The 35S expression cassette is flanked by two Not I restriction sites which permits cloning into binary expression vectors, such as pART27 which contains a unique NotI site. Its construction as described in Gleave, AP (1992), a map is shown in Figure 43.
Plasmid pART7.35S.SCBV.cass
Plasmid p35S.CMV.cass was designed to express two separate gene sequences cloned into a single plasmid. To create this plasmid, sequences corresponding to the nos terminator and the SCBV promoter were amplified by PCR then cloned in the polylinker of pART7 between the 35S promoter and OCS. - 57 -
The resulting plasmid has the following arrangement of elements:
35S promoter - polylinker 1 - NOS terminator - SCBV promoter - polylinker 2 - OCS terminator.
Expression of sequences cloned into polylinker 1 is controlled by the 35S promoter, expression of sequences cloned into polylinker 2 is controlled by the SCBV promoter.
The NOS terminator sequences were amplified from the plasmid pAHC27 (Christensen and Quail, 1996) using the two oligonucleotides;
NOS 5' (forward primer; SEQ ID ??) 5'-GGATTCCCGGGACGTCGCGAATTTCCCCCGATCGTTC-3'; and
NOS 3' (reverse primer; SEQ ID ??)
5'-CCATGGCCATATAGGCCCGATCTAGTAACATAG-3'
Nucleotide residues 1 to 17 for NOS 5' and 1 to 15 for NOS 3' represent additional nucleotides designed to assist in construct preparation by adding additional restriction sites. For NOS 5' these are BamHl, Smal, Aatll and the first 4 bases of an Nrul site, for NOS 3' these are Ncol and Sfil sites. The remaining sequences for each oligonucleotide are homologous to the 5' and 3' ends respectively of NOS sequences in pAHC 27.
The SCBV promoter sequences were amplified from the plasmid pScBV-20 (Tzafir et al, 1998) using the two oligonucleotides:
SCBV 5': 5'-CCATGGCCTATATGGCCATTCCCCACATTCAAG-3'; and
SCBV 3': 5'-AACGTTAACTTCTACCCAGTTCCAGAG-3' 58
Nucleotide residues 1 to 17 of SCBV 5' encode Ncol and Sfil restriction sites designed to assist in construct preparation, the remaining sequences are homologous to upstream sequences of the SCMV promoter region. Nucleotide residues 1 to 9 of SCBV 3' encode Psp10461 and Hpal restriction sites designed to assist in construct preparation, the remaining sequences are homologous to the reverse and complement of sequences near the transcription initiation site of SCBV.
Sequences amplified from pScBV-20 using PCR and cloned into pCR2.1 (Invitrogen) to produce pCR.NOS and pCR.SCBV respectively. Smal l/Sfil cut pCR.NOS and Sfil/Hpal cut pCR.SCBV were ligated into Sma I cut pART7 and a plasmid with a suitable orientation was chosen and designated pART7.35S.SCBV.cass, a map of this construct is shown in Figure 43.
4. Intermediate constructs Plasmid pBC.PVY
A region of the PVY genome was amplified by PCR using reverse-transcribed RNA isolated from PVY-infected tobacco as a template using standard protocols and cloned into a plasmid pGEM 3 (Stratagene), to create pGEM.PVY. A Sall/Hindlll fragment from pGEM.PVY, corresponding to a Sall/Hindlll fragment positions 1536-2270 of the PVY strain O sequence (Ace. No D12539, Genbank), was then subcloned into the plasmid pBC (Stratagene Inc.) to create pBC.PVY (Figure 44).
Plasmid pSP72.PVY
Plasmid pSP72.PVY was prepared by inserting an EcoRI/Sall fragment from pBC.PVY into EcoRI/Sall cut ρSP72 (Promega). This construct contains additional restriction sites flanking the PVY insert which were used to assist subsequent manipulations. A map of this construct is shown in Figure 45.
Plasmid ClapBC.PVY Plasmid Cla pBC.PVY was prepared by inserting a Clal/Sall fragment from pSP72.PVY - 59 -
into Clal/Sal I cutpBC (Stratagene). This construct contains additional restriction sites flanking the PVY insert which were used to assist subsequent manipulations. A map of this construct is shown in Figure 46.
Plasmid pBC.PVYx2
Plasmid pBC.PVYx2 contains two direct head-to-tail repeats of the PVY sequences derived from pBC.PVY. The plasmid was generated by cloning an Accl/Clal PVY fragment from pSP72.PVY into Accl cut pBC.PVY and is shown in Figure 47.
Plasmid pSP72.PVYx2
Plasmid pSP72.PVYx2 contains two direct head-to-tail repeats of the PVY sequences derived from pBC.PVY. The plasmid was generated by cloning an Accl/Clal PVY fragment from pBc.PVY into Accl cut pSP72.PVY and is shown in Figure 48.
Plasmid pBC.PVYx3
Plasmid pBC.PVYx3 contains three direct head-to-tail repeats of the PVY sequences derived from pBC.PVY. The plasmid was prepared by cloning an Accl/Clal PVY fragment from pSP72.PVY into Accl cut pBC.PVYx2 and is shown in Figure 49.
Plasmid pBC.PVYx4
Plasmid pBC.PVYx4 contains four direct head-to-tail repeats of the PVY sequences derived from pBC.PVY. The plasmid was prepared by cloning the direct repeat of PVY sequences from pSP72.PVYx2 as an Accl/Clal fragment into Accl cut pBC.PVYx2 and is shown in Figure 50.
Plasmid pBC.PVY.LNYV
All attempts to create direct palindromes of PVY sequences failed, presumably such sequence arrangements are unstable in commonly used E. coli cloning hosts. Interrupted palindromes however proved stable. 60 -
To create interrupted palindromes of PVY sequences a "stuffer" fragment of approximately 360 bp was inserted into Cla pBV.PVY downstream of the PVY sequences. The stuffer fragment was made as follows:
A clone obtained initially from a cDNA library prepared from lettuce necrotic yellows virus (LNYV) genomic RNA (Deitzgen etal, 1989), known to contain the 4b gene of the virus, was amplified by PCR using the primers:
LNYV 1:5'-ATGGGATCCGTTATGCCAAGAAGAAGGA-3'; and
LNYV 2:5'-TGTGGATCCCTAACGGACCCGATG-3'
The first 9 nucleotide of these primers encode a BamHl site, the remaining nucleotides are homologous to sequences of the LNYV 4b gene.
Following amplification, the fragment was cloned into the EcoRI site of pCR2J (Stratagene). This EcoRI fragment was cloned into the EcoRI site of Cla pBC.PVY to create the intermediate plasmid pBC.PVY.LNYV which is shown in Figure 51.
Plasmid pBC.PVY.LNYV.PVY
The plasmid pBC.PVY. LNYV. YVP contains an interrupted direct repeat of PVY sequences, to create this plasmid a Hpal/Hincll fragment from pSP72 was cloned into Smal-digested pBC.PVY.LNYV and a plasmid containing the sense orientation isolated, a map of this construct is shown in Figure 52.
Plasmid pBC.PVY.LNYV.YVPΔ
The plasmid PBV.PVY.LNYV.YVPΔ contains a partial interrupted palindrome of PVY sequences. One arm of the palindrome contains all the PVY sequences from pBC.PVY, the other arm contains part of the sequences from PVY, corresponding to sequences between the EcoRV and Hindi sites of pSP72.PVY. To create this plasmid 61 -
an EcoRV/Hincll fragment from pSP72.PVY was cloned into Smal-digested pBC.PVY.LNYV and a plasmid containing the desired orientation isolated, a map of this construct is shown in Figure 53.
Plasmid pBC.PVY.LNYV. YVP The plasmid pBC.PVY.LNYV.YVP contains an interrupted palindrome of PVY sequences. To create this plasmid a Hpal/Hincll fragment from pSP72. was cloned into Sma-digested pBC.PVY.LNYV and a plasmid containing the antisense orientation isolated, a map of this construct is shown in Figure 54.
5. Control plasmids
Plasmids pART7.PVY & pART7.PVY
Plasmid pART7.PVY (Figure 55) was designed to express PVY sequences driven by the 35S promoter. This plasmid serves as a control construct in these experiments, against which all other constructs was compared. To generate this plasmid a Clal/Accl fragment from ClapBC.PVY was cloned into Clal-digested pART7 and a plasmid with expected to express a sense PVY sequence with respect to the PVY genome, was selected. Sequences consisting of the 35S promoter, PVY sequences and the OCS terminator were excised as a NotI fragment and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.
Plasmids pART7.35S.PVY.SCBV.O & pART27.35S.PVY.SCBV.O
Plasmid pART7.35S.PVY.SCBV.O (Figure 56) was designed to act as a control for co- expression of multiple constructs from a single plasmid in transgenic plants. The 35S promoter was designed to express PVY sense sequences, whilst the SCBV promoter was empty. To generate this plasmid, the PVY fragment from Cla pBC.PVY was cloned as a Xhol/EcoRI fragment into Xhol/EcoRI-digested pART7.35S.SCBV.cass to create p35S.PVY.SCBV>O. Sequences consisting of the 35S promoter driving sense PVY sequences and the NOS terminator and the SCBV promoter and OCS terminator were excised as a NotI fragment and cloned into pART27, a plasmid with 62
the desired insert orientation was isolated and designated pART27.35S.PVY.SCBV.O.
Plasmids pART7.35S.O.SCBV.PVY & pART27.35S.O.SCBV.PVY
Plasmid pART27.35S.O.SCBV.PVY (Figure 57) was designed to act as an additional control for co-expression of multiple constructs from a single plasmid in transgenic plants. No expressible sequences were cloned behind the 35S promoter, whilst the SCBV promoter drove expression of a PVY sense fragment. To generate this plasmid, the PVY fragment from Cla pBC.PVY was cloned as a Clal fragment into Clal-digested pART7.35S.SCBV.cass, a plasmid containing PVY sequences in a sense orientation was isolated and designated p35S.O.SCBV.PVY. Sequences, consisting of the 35S promoter and NOS terminator, the SCBV promoter driving sense PVY sequences and the OCS terminator were excised as a NotI fragment and cloned into pART27, a plasmid with the desired insert orientation was isolated and designated pART27.35S.O.SCBV.PVY.
Plasmids pART7.35S.O.SCBV.YVP & pART7.35S.O.SCBV.YVP Plasmid pART7.35S.O.SCBV.YVP (Figure 58) was designed to act as an additional control for co-expression of multiple constructs from a single plasmid in transgenic plants. No expressible sequences were cloned behind the 35S promoter, whilst the SCBV promoter drove expression of a PVY antisense fragment. To generate this plasmid, the PVY fragment from Cla pBC.PVY was cloned as a Clal fragment into Clal- digested p35S.SCBV.cass, a plasmid containing PCY sequences in an antisense orientation was isolated and designated p35S.O.SCBV.YVP. Sequences, consisting of the 35S promoter and NOS terminator, the SCBV promoter driving sense PVY sequences and the OCS terminator were excised as a NotI fragment and cloned into pART27, a plasmid with the desired insert orientation was isolated and designated pART27.35S.O.SCBV.YVP.
6. Test plasmids Plasmids pART7.PVYx2 & pART27.PVYx2 - 63 -
Plasmid pART7.PVYx2 (Figure 59) was designed to express a direct repeat of PVY sequences driven by the 35S promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx2 were cloned as a Xhol/BamHI fragment into Xhol/BamHI cut pART7. Sequences consisting of the 35 S promoter, direct repeats of PVY and the OCS terminator were excised as a NotI fragment from pART7.PVYx2 and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.PVYx2.
Plasmids pART7.PVYx3 & pART27.PVYx3 Plasmid pART7.PVYx3 (Figure 60) was designed to express a direct repeat of three PVY sequences driven by the 35S promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx3 were cloned as a Xhol/BamHI fragment into Xhol/BamHI cut pART7. Sequences consisting of the 35S promoter, direct repeats of PVY and OCS terminator were excised as a NotI fragment from pART.PVYx3 and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.PVYx3.
Plasmids pART7.PVYx4 & pART27.PVYx4
Plasmid pART7.PVYx4 (Figure 61) was designed to express a direct repeat of four PVY sequences driven by the 35S promoter in transgenic plants. To generate this plasmid, direct repeats from pBC.PVYx4 were cloned as a Xhol/BamHI fragment into xhol/BamHI cut pART7. Sequences consisting of the 35S promoter, direct repeats of PVY and the OCS terminator were excised as a NotI fragment from pART7.PVYx3 and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27.PVYx3.
Plasmids pART7.PVY.LNYV.PVY & pART27.PVY.LNYV.PVY
Plasmid pART7. PVY. LNYV. PVY (Figure 62) was designed to express the interrupted direct repeat of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the interrupted direct repeat of PVY from 64 -
pBC.PVY.LNYV.PVY as a Xhol/Xbal fragment into pART7 digested with Xhol/Xbal. Sequences consisting of the 35S promoter, the interrupted direct repeat of PVY sequences and the OCS terminator were excised from pART7.PVY.LNYV.PVY as a NotI fragment and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27. PVY. LNYV. PVY.
Plasmids pART7.PVY.LNYV.YVPΔ & pART27.PVY.LNYV.YVPΔ
Plasmid pART7. PVY. LNYV. YVPΔ (Figure 63) was designed to express the partial interrupted palindrome of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the partial interrupted palindrome of PVY sequences from pBC.PVY. LNYV. YVPΔ as a Xhol/Xbal fragment into pART7 digested with Xhol/Xbal. Sequences consisting of the 35S promoter, the partial interrupted palindrome of PVY sequences and the OCS terminator were excised from pART7. PVY. LNYV. YVPΔ as a NotI fragment and cloned into Notl-digested pART27, a plasmid with the desired insert orientation was selected and designated pART27. PVY. LNYV. YVP.
Plasmids pART7.PVY.LNYV.YVP & pART27.PVY.LNYV. YVP
Plasmid pART7. PVY. LNYV. YVP (Figure 64) was designed to express the interrupted palindrome of PVY sequences driven by the 35S promoter in transgenic plants. This construct was prepared by cloning the interrupted palindrome of PVY sequences from PBC.PVY.LNYV.YVPΔ as a Xhol/Xbal fragment into pART7 digested with Xhol/Xbal. Sequences consisting of the 35S promoter, the interrupted palindrome of PVY sequences and the OCS terminator were excised from pART7.PVY.LNYV. YVP as a NotI fragment and cloned into ρART27, a plasmid with the desired insert orientation was selected and designated pART27. PVY. LNYV. YVP.
Plasmids pART7.35S.PVY.SCBV.YVP & pART27.35S.PVY.SCBV.YVP
Plasmid pART7.35S. PVY. SCBV. YVP (Figure 65) was designed to co-express sense and antisense constructs in transgenic plants. To generate this plasmid the PVY 65
fragment from Cla pBC.PVY was cloned as a Xhol/EcoRI fragment into xhol/EcoRI- digested p35S.SCBV.O.SCBV.YVP. Sequences, consisting of the 35S promoter driving sense PVY sequences and the NOS terminator and the SCBV promoter driving antisense PVY and the OCS terminator were excised as a NotI fragment and cloned into pART27, a plasmid with the desired insert orientation was isolated and designated pART27.35S.PVY.SCBV. YVP.
Plasmids pART7.35S.PVYx3.SCBV.YVPx3 & pART27.35S.PVYx3.SCBV.YVPx3
Plasmid pART7.35S.PVYx3.SCBV.YVPx3 (Figure 66) was designed to co-express sense and antisense repeats of PVY in transgenic plants, to generate this plasmid, the intermediate pART7.35S.O.SCBV.YVPx3 was constructed by cloning the triple direct PVY repeat from ClapBC.PVYx3 as a Clal/Accl fragment into Cla-digested p35S.SCBV.cass and isolating a plasmid with an antisense orientation, for p35S.PVYx3.SCBV.YVPx3 the triple direct PVY repeat from Cla pBC.PVYx3 was cloned as a KpnI/Smal fragment into KpnI/Smal-digested p35S.O.SCBV.YVPx3 to create p35S.PVYx3.SCBV.YVPx3. Sequences including both promoters, terminators and direct PVY repeats were isolated as a NotI fragment and cloned into pART27. A plasmid with an appropriate orientation was chosen and designated pART27.35S.PVYx3.SCBV.
Plasmids pART7.PVYx3.LNYV.YVPx3 & pART27.PVYx3.LNYV.YVPx3 Plasmid pART7.PVYx3.LNYV.YVPx3 (Figure 67) was designed to express triple repeats of PVY sequences as an interrupted palindrome. To generate this plasmid an intermediate, pART7x3. PVY. LNYV. YV was constructed by cloning a PVY.LNYV.YVP fragment from pBC.PVY. LNYV. YVP as an Accl/Clal fragment into the plasmid pART7.PVYx2. pART7.35S.PVYx3.LNYV.YVPx3, was made by cloning an additional PVY direct repeat from pBC.PVYx2 as an Accl/Clal fragment into Clal digested pART7x3.PVY.LNYV.YVP. Sequences from pART7.35S.PVYx3.LNYV.YVPx3, including the 35S promoter, all PVY sequences and the OCS terminator were excised as a NotI fragment and cloned into Notl-digested pART27, a plasmid with an - 66 -
appropriate orientation was chosen and designated pART27.35S.PVYx3.LNYV.
Plasmids pART7.PVY multi & pART27.PVY multi
Plasmid pART7.35S.PVY multi (Figure 68) was designed to express higher order direct repeats of regions of PVY sequences in transgenic plants. Higher order direct repeats of a 72 bp of the PVY Nia region from PVY were prepared by annealing two partially complementary oligonucleotides as follows:
PVY1 : 5'-TAATGAGGATGATGTCCCTACCTTTAATTGGCAGAAATTTCTGTGGAAAGACAG GGAAATCTTTCGGCATTT-3'; and
PVY2: δ'-TTCTGCCAATTAAAGGTAGGGACATCATCCTCATTAAAATGCCGAAAGATT TCCCTGTCTTTCCACAGAAAT-3'
The oligonucleotides were phosphorylated with T4 polynucleotide kinase, heated and cooled slowly to permit self-annealing, ligated with T4 DNA ligase, end-filled with Klenow polymerase and cloned into pCR2.1 (Invitrogen). Plasmids containing multiple repeats were isolated and sequences were cloned as EcoRI fragments in a sense orientation into EcoRI-digested pART7, to create the intermediate pART7.PVY multi. to create pART27.PVY multi, the 35S promoter, PVY sequences and the OCS terminator were excised as a NotI fragment and cloned into Notl-digested pART27. A plasmid with an appropriate insert orientation was isolated and designated pART27. PVY multi. - 67 -
EXAMPLE 6 Inactivation of virus gene expression in mammals
Viral immune lines are created by expressing viral sequences in stably transformed cell lines.
In particular, lytic viruses are used for this approach since cell lysis provides very simple screens and also offer the ability to directly select for potentially rare transformation events which might create viral immunity. Sub-genomic fragments derived from a simple single stranded RNA virus (Bovine enterovirus - BEV) or a complex double stranded DNA virus, Herpes Simplex Virus I (HSV I) are cloned into a suitable vector and expressed in transformed cells. Mammalian cell lines are transformed with genetic constructs designed to express viral sequences driven by the strong cytomegalovirus (CMV-IE) promoter. Sequences utilised include specific viral replicase genes. Random "shotgun" libraries comprising representative viral gene sequences, may also be used and the introduced dispersed nucleic acid molecule, to target the expression of virus sequences.
Exemplary genetic constructs for use in this procedure, comprising nucleotide sequences derived from the BEV RNA-dependent RNA polymerase gene, are presented herein.
For viral polymerase constructs, large numbers (approximately 100) of transformed cell lines are generated and infected with the respective virus. For cells transformed with shotgun libraries very large numbers (hundreds) of transformed lines are generated and screened in bulk for viral immunity. Following virus challenge, resistant cell lines are selected and analysed further to determine the sequences conferring immunity thereon.
Resistant cell lines are supportive of the ability of the introduced nucleotide sequences to inactivate viral gene expression in a mammalian system. - 68 -
Additionally, resistant lines obtained from such experiments are used to more precisely define molecular and biochemical characteristics of the modulation which is observed.
EXAMPLE 8
Induction of virus resistance in transgenic plants
Agrobacterium tumefaciens, strain LBA4404, was transformed independently with the constructs pART27.PVY, pART27.PVYx2, pART27.PVYx3, pART27.PVYx4, pART27. PVY. LNYV. PVY, pART27. PVY. LNYV. YVPΔ, pART27.PVY.LNYV.YVP, pART27.35S.PVY.SCBV.O, pART27.35S.O.SCBV.PVY, pART27.35S.O.SCBV.YVP, PART27.35S . PVY. SCBV. YVP, pART27.35S. PVYx3.SCBV.YPVx3, pART27.PVYx3.LNYV.YVPx3 and pART27.PVYx10, using tri-parental matings. DNA mini-preps from these strains were prepared and examined by restriction with NotI to ensure they contained the appropriate binary vectors.
Nicotiana tabaccum (cultivar W38) were transformed with these Agrobactehum strains using standard procedures. Putative transformed shoots were excised and rooted on media containing kanamycin. Under these conditions we have consistently observed that only transgenic shoots will root on kanamycin plates. Rooted shoots were transferred to soil and allowed to establish. After two to three weeks, vigorous plants with at least three sets of leaves were chosen and infected with PVY.
Viral inoculum was prepared from W38 tobacco previously infected with the virus, approximately 2 g of leaf material, showing obvious viral symptoms were ground with carbarundum in 10 ml of 100mM Na phosphate buffer (pH 7.5). the inoculum was diluted to 200 ml with additional Na phosphate buffer. Two leaves from each transgenic plant were sprinkled with carbarundum, then 0.4 ml of inoculum was applied to each leaf and leaves rubbed fairly vigorously with fingers. Using this procedure 100% of non-transgenic control plants were infected with PVY. 69
To assay for viral resistance and immunity transgenic plants are monitored for symptom development. The PVY strain (PVY-D, an Australian PVY isolate) gives obvious symptoms on W38 tobacco, a vein clearing symptom is readily observed on the two leaves above the inoculated leaves, subsequent leaves show uniform chlorotic lesions. Symptom development was monitored over a six week period.
Transgenic lines were described as resistant if they showed reduced viral symptoms, which manifests as a reduction in the leaf are showing chlorotic lesions. Resistance ranges from very strong resistance where only a few viral lesions are observed on a plant to weak resistance which manifects as reduced symptoms on leaves that develop late in plant growth.
Transgenic plants which showed absolutely no evidence of viral symptoms were classified as immune. To ensure these plants were immune they were re-inoculated with virus, most plants remained immune, the few that showed symptoms were re- classified as resistant.
For plant lines generated Southern blots are performed, resistance in subsequent generations is monitored to determine that resistance/immunity is transmissable. Additionally, the breadth of viral resistance is monitored by challenging lines with other PVY strains, to determine whether host range susceptibility is modified.
Results from these experiments are described in Table 2 . These data indicate that constructs comprising tandem repeats of target gene sequence, either in the configuration of palindromes, interrupted palindromes as direct repeat sequences, are capable of conferring viral resistance and/or immunity in transgenic plants.
Accordingly, such inverted and/or direct repeat sequences modulate expression of the virus target gene in the transgenic plant. - 70 -
Constructs combining the use of direct and inverted repeat sequences, namely pART27.35S.PVYx3.SCBV.YVPx3 and pART27.PVYx3.LNYV.YVPx3, are also useful in modulating gene expression.
EXAMPLE 9
Inactivation of Gait in animal cells
To assay for Gait inactivation, porcine PK2 cells were transformed with the relevant constructs. PK2 cells constitutively express Gait enzyme, the activity of which results in the addition of a variety of α-1 ,3-galactosyl groups to a range of proteins expressed on the cell surface of these cells. Cells were transformed using lipofectin and stably transformed lines were selected using genetecin.
As an initial assay cell lines were probed for the presence of the Gait-encoded epitope, i.e. α-1 ,3-galactosyl moieties decorating cell surface proteins, using the lectin IB4. IB4 binding was assayed either in situ or by FACS sorting.
For in situ binding, cells were fixed to solid supports with cold methanol for 5 mins, cells were rinsed in PBS (phosphate buffered saline) and non-specific IB4 binding was blocked with 1% BSA in PBS for 10 mins. Fixed cells were probed using 20 ug/ml IB4- biotin (Sigma) in 1% BSA, PBS for 30 mins at room temperature, cells were washed in PBS then probed with a 1 :200 dilution of ExtrAvidin-FITC (Sigma) in PBS for 30 mins followed by further rinses in PBS. Cells were then examined using fluorescence microscopy, under these conditions the outer surface of PK2 control cells uniformly stained green.
For FACS analysis, cells were suspended after treatment with trypsin, washed in HBSS/Hepes (Hank's buffered saline solution with 20 mM Hepes, pH7.4) and probed with 10 ug/ml IB4-biotin (Sigma) in HBSS/Hepes for 45 mins at 4°C. Cells were washed in HBSS/Hepes, probed with a 1:200 dilution of ExtrAvidin-FITC (Sigma) in HBSS/Hepes for 45 mins at 4°C at and rinsed in cold HBSS/Hepes prior to FACS - 71 -
sorting.
Using this approach transformed cell lines are assayed for Gait inactivation and quantitative assessment of construct effectiveness is determined. Moreover cell lines showing Gait inactivation are isolated and subject to further molecular analyses to determine the mechanism of gene inactivation.
PLASMID CONSTRUCT No. OF PERCENTAGE OF PLANTS SHOWING o PLANTS SPECIFIED PHENOTYPE TESTED SUSCEPTIBLE IMMUNE RESISTANT
pART27.PVY 19 16 1 2 pART27.PVYx2 13 5 4 4 pART27.PVYx3 21 2 5 14
5 pART27.PVYx4 21 5 7 9 r\_
pART27.35S.PVY.SCBC.O 25 8 0 17 pART27.35S.O.SCBV.PVY 22 8 0 14 pART27.35S.O.SCBV.YVP 18 14 0 4
0 pART27.35S.PVY.SCBV. YVP 17 3 8 6
o pART27.PVY.LNYV.PVY 26 18 2 6 C pART27. PVY. LNYV. YVP 20 6 10 vo
4 \o o o >-* pART27. PVY. LNYV. YVPΔ 18 7 11 0 SO
Figure imgf000074_0001
w
- 73
REFERENCES
1. An et al. (1985) EMBO J 4:277-284.
2. Armstrong, et al.Plant Cell Reports 9: 335-339, 1990.
3. Ausubel, F.M. et a/.(1987) In: Current Protocols in Molecular Biology, Wiley 5 Interscience (ISBN 047140338)..
4. Chalfie.M. et al (1994) Science 263: 802-805.
5. Christensen, A.H. and Quail, P.H. (1996) Transgenic Research 5: 213-218.
6. Christou, P., et al. Plant Physiol 87: 671-674, 1988.
7. Cormack, B. et al (1996) Gene 173: 33-38.
10 8. Crossway et al., Mol. Gen. Genet. 202:179-185, 1986.
9. Dorer, D.R., and Henikoff, S. (1994) Cell 7: 993-1002.
10. Fromm et al. Proc. Natl. Acad. Sci. (USA) 82:5824-5828, 1985.
11. Gleave, A. P. (1992) Plant Molecular Biology 20:1203-1207.
12. Hanahan, D. (1983) J. Mol.Biol. 166: 557-560.
15 13. Herrera-Estella et al., Nature 303: 209-213, 1983a.
14. Herrera-Estella et al.,EMBO J. 2: 987-995, 1983b.
15. Herrera-Estella et al. In: Plant Genetic Engineering, Cambridge University Press, N.Y., pp 63-93, 1985.
16. Inouye, S. and Tsuji, F.I. (1994) FEBS Letts. 341: 277-280. 20 17. Jackson, I.J. (1995) Ann. Rev. Genet. 28: 189-217.
18. Krens, F.A., et al., Nature 296: 72-7 'A, 1982.
19. Kwon, B.S. et al. (1988) Biochem. Biophys. Res. Comm. 153:1301- 1309.
20. Pal-Bhadra, M. et al. (1997) Cell 90: 479-490.
21. Paszkowski et al. , EMBO J. 3:2717-2722 , 1984 25 22. Prasher, D.C. et al. (1992) Gene 111: 229-233.
23. Sanford, J.C, et al., Particulate Science and Technology 5: 27-37, 1987.

Claims

- 74CLAIMS
1. A method of repressing, delaying or otherwise reducing the expression of a target gene in a cell, tissue or organ, said method comprising introducing to said cell, tissue or organ one or more dispersed nucleic acid molecules or foreign nucleic acid molecules comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or complementary thereto for a time and under conditions sufficient for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
2. The method according to claim 1 wherein the dispersed nucleic acid molecules or foreign nucleic acid molecules comprise inverted repeats of the target gene sequence or a region thereof or complementary thereto.
3. The method according to claim 1 wherein the dispersed nucleic acid molecules or foreign nucleic acid molecules comprise direct repeats of the target gene sequence or a region thereof or complementary thereto.
4. The method according to claim 1 wherein the dispersed nucleic acid molecules or foreign nucleic acid molecules comprise both direct and inverted repeats of the target gene sequence or a region thereof or complementary thereto.
5. The method according to any one of claims 1 to 4, wherein the number of copies of the target gene sequence or region thereof or complementary thereto in the dispersed nucleic acid molecule or foreign nucleic acid molecule is two.
6. The method according to any one of claims 1 to 4, wherein the number of copies of the target gene sequence or region thereof or complementary thereto in the dispersed nucleic acid molecule or foreign nucleic acid molecule is three. - 75 -
7. The method according to any one of claims 1 to 4, wherein the number of copies of the target gene sequence or region thereof or complementary thereto in the dispersed nucleic acid molecule or foreign nucleic acid molecule is four.
8. The method according to any one of claims 1 to 4, wherein the number of copies of the target gene sequence or region thereof or complementary thereto in the dispersed nucleic acid molecule or foreign nucleic acid molecule is six.
9. The method according to any one of claims 1 to 4, wherein the number of copies of the target gene sequence or region thereof or complementary thereto in the dispersed nucleic acid molecule or foreign nucleic acid molecule is ten.
10. The method according to any one of claims one to 9 wherein the dispersed nucleic acid molecule or foreign nucleic acid molecule comprises tandem repeats of the target gene sequence and wherein one or more of the repeated units of said tandem repeats is separated from another unit be a nucleic acid-containing stuffer fragment.
11. The method according to any one of claims 1 to 10 wherein the cell, tissue or organ is an animal cell, tissue or organ.
12. The method according to claim 11 wherein the animal is a mouse.
13. The method according to any one of claims 1 to 10 wherein the cell, tissue or organ is a plant cell, tissue or organ.
14. The method according to claim 13 wherein the plant is a tobacco plant.
15. The method according to any one of claims 1 to 14 wherein the target gene is a gene which is contained within the genome of the cell, tissue or organ. - 76 -
16. The method according to claim 15 wherein the target gene is ╬▒-1 , 3- galactosyltransferase.
17. The method according to any one of claims 1 to 14 wherein the target gene is 5 derived from the genome of a pathogen of the cell, tissue or organ or an organism comprising said cell, tissue or organ.
18. The method according to claim 17 wherein the pathogen is a virus.
10 19. The method according to claim 18 wherein the virus is an animal pathogen.
20. The method according to claim 19 wherein the virus is BEV.
21. The method according to claim 18 wherein the virus is a plant pathogen. 15
22. The method according to claim 21 wherein the virus is PVY.
23. The method according to any one of claims 1 to 22 further comprising selecting the dispersed nucleic acid molecule(s) or foreign nucleic acid molecule(s) according 0 to their ability to effectively modulate expression of the target gene.
24. A method of repressing, delaying or otherwise reducing the expression of a target gene in a cell, tissue or organ, said method comprising:
(i) selecting one or more dispersed nucleic acid molecules or foreign nucleic 5 acid molecules which comprise tandem repeats of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a region thereof or which is complementary thereto;
(ii) producing a synthetic gene comprising said dispersed nucleic acid molecules or foreign nucleic acid molecules operably connected to a promoter 30 sequence operable in said cell, tissue or organ; 77
(iii) introducing said synthetic gene to said cell, tissue or organ; and (iv) expressing said synthetic gene in said cell, tissue or organ for a time and under conditions sufficient for translation of the mRNA product of said target gene to be modified, subject to the proviso that the transcription of said mRNA 5 product is not exclusively repressed or reduced.
25. A method of conferring resistance or immunity to a viral pathogen upon a cell, tissue, organ or whole organism, comprising introducing one or more dispersed nucleic acid molecules or foreign nucleic acid molecules which comprise tandem repeats of 10 a nucleotide sequence derived from the viral pathogen or a complementary sequence thereto for a time and under conditions sufficient for translation of the mRNA product of a virus gene to be delayed or otherwise reduced, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
15 26. The method according to claim 25 wherein the viral pathogen is a plant pathogen.
27. The method according to claim 26 wherein the virus is PVY.
20 28. The method according to claim 25 wherein the virus is an animal pathogen.
29. The method according to claim 28 wherein the virus is BEV.
30. The method according to any one of claims 25 to 29 further comprising selecting 25 the dispersed nucleic acid molecule(s) or foreign nucleic acid molecule(s) according to their ability to confer resistance or immunity on the cell, tissue, organ or organism.
31. A method of conferring resistance or immunity to a viral pathogen upon a cell, tissue, organ or whole organism, comprising:
30 (i) selecting one or more dispersed nucleic acid molecules or foreign nucleic 78
acid molecules which comprise tandem repeats of a nucleotide sequence derived from the viral pathogen or a complementary sequence thereto; (ii) producing a synthetic gene comprising said dispersed nucleic acid molecules or foreign nucleic acid molecules operably connected to a promoter sequence operable in said cell, tissue, organ or whole organism;
(iii) introducing said synthetic gene to said cell, tissue, organ or whole organism; and
(iv) expressing said synthetic gene in said cell, tissue or organ for a time and under conditions sufficient for translation of the mRNA product of a gene of the virus to be modified, subject to the proviso that the transcription of said mRNA product is not exclusively repressed or reduced.
32. The method according to any one of claims 25 to 31 , wherein the dispersed nucleic acid molecules or foreign nucleic acid molecules comprise multiple copies of nucleotide sequence encoding a viral replicase, polymerase, coat protein or uncoating gene.
33. The method according to claim 32 wherein the dispersed nucleic acid molecules or foreign nucleic acid molecules comprise multiple copies of nucleotide sequence encoding a viral polymerase.
34. The method according to claim 32 wherein the dispersed nucleic acid molecules or foreign nucleic acid molecules comprise multiple copies of nucleotide sequence encoding a viral coat protein.
35. A synthetic gene which is capable of repressing, delaying or otherwise reducing the expression of a target gene in a cell, tissue, organ or whole organism, wherein said synthetic gene comprises a dispersed nucleic acid molecule or a foreign nucleic acid molecule comprising multiple copies of a nucleotide sequence which is substantially identical to the nucleotide sequence of said target gene or a derivative thereof or a - 79 -
complementary sequence thereto placed operably under the control of a promoter sequence which is operable in said cell, tissue, organ or whole organism.
36. The synthetic gene according to claim 35, wherein the dispersed nucleic acid 5 molecule or a foreign nucleic acid molecule comprises tandem inverted and/or direct repeats of a genetic sequence that is endogenous to the genome of the cell, tissue, organ or organism or which is derived from a non-endogenous gene of the cell, tissue, organ or organism.
10 37. The synthetic gene according to claim 36 wherein the non-endogenous gene is derived from a viral pathogen of the cell, tissue, organ or organism.
38. The synthetic gene according to claim 37 wherein the non-endogenous gene is derived from an animal virus.
15
39. The synthetic gene according to claim 38 wherein the animal virus is BEV.
40. The synthetic gene according to claim 38 wherein the non-endogenous gene is derived from the BEV polymerase gene. 0
41. The synthetic gene according to claim 40 wherein the promoter is the CMV-IE promoter or SV40 promoter sequence.
42. The synthetic gene according to claim 37 wherein the non-endogenous gene 5 is derived from a plant virus.
43. The synthetic gene according to claim 42 wherein the plant virus is PVY.
44. The synthetic gene according to claim 43 wherein the promoter is the CaMV 30 35S promoter or the SCBV promoter sequence. 80
45. The synthetic gene according to claims 35 or 36 wherein the dispersed nucleic acid molecule or a foreign nucleic acid molecule comprises tandem inverted and/or direct repeats of the porcine ╬▒-1 ,3-galactosyltransferase gene.
5 46. The synthetic gene according to claim 45 wherein the porcine ╬▒-1, 3- galactosyltransferase gene is placed operably in connection with the CMV promoter sequence.
47. The synthetic gene according to any one of claims 35 to 46 wherein the multiple 10 copies of the nucleotide sequence of the target gene are operably connected to two or more promoter sequences.
48. The synthetic gene according to claim 47 wherein each of the multiple copies of the nucleotide sequence of the target gene are operably connected to spatially
15 separate promoter sequences.
49. A genetic construct comprising the synthetic gene according to any one of claims 35 to 48.
20 50. The genetic construct according to claim 49 selected from the list comprising plasmid pCMV.BEVx2; plasmid pCMV.BEV.GFP.VEB; plasmid pCMV.BEV.SV40L.BEV; and plasmid pCMV.BEV.SV40L.VEB.
51. The genetic construct according to claim 49 selected from plasmid 25 pCMV.Galtx2; and pCMV.Galtx4.
52. The genetic construct according to claim 49 selected from the list comprising plasmid pSP72.PVYx2; plasmid pBC.PVYx2; plasmid pBC.PVYx3; plasmid pBC.PVYx4; plasmid pART27.PVYx2; plasmid pART27.PVYx3; plasmid
30 pART27.PVYx4; plasmid PBC.PVY.LNYV.YVPΔ; plasmid pBC.PVY.LNYV.YVP; 81 -
plasmid pBC.PVY.LNYV.PVY; plasmid pART27. PVY. LNYV. PVY; plasmid PART27.PVY.LNYV.YVPΔ; plasmid pART27.PVY.LNYV.YVP; plasmid pART27.35S.PVY.SCBV. YVP; plasmid pART27.35S.PVYx3.SCBV.YVPx3; plasmid pART27.PVYx3.LNYV.YVPx3; and plasmid pART27.PVYx10. 5
53. Use of the genetic construct according to claim 50 to confer immunity or resistance against BEV upon an animal cell, tissue or organ or a whole animal.
54. Use of the genetic construct according to claim 51 to delay, repress or otherwise 10 reduce expression of ╬▒-1 ,3-galactosyltransferase in a cell, tissue, organ or whole organism that would otherwise express same.
55. Use of the genetic construct according to claim 52 to confer immunity or resistance against PVY upon a plant cell, tissue, organ or whole plant.
15
56. Use according to claim 55, wherein the plant is tobacco.
57. A cell, tissue, organ or whole organism comprising the synthetic gene according to any one of claims 35 to 48 or the genetic construct according to any one of claims
20 49 to 52 .
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HU0101225A HU230353B1 (en) 1998-03-20 1999-03-19 Control of gene expression
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BRPI9908967A BRPI9908967B1 (en) 1998-03-20 1999-03-19 processes for suppressing, retarding or otherwise reducing expression of a target gene in a plant cell
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SK1372-2000A SK287538B6 (en) 1998-03-20 1999-03-19 Control of gene expression
NZ506648A NZ506648A (en) 1998-03-20 1999-03-19 Control of gene expression through introduction of synthetic tandem repeats to reduce translation of mRNA
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US10/646,070 US7754697B2 (en) 1998-03-20 2003-08-22 Control of gene expression
US11/218,999 US8168774B2 (en) 1998-03-20 2005-09-02 Control of gene expression
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Cited By (224)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000068374A1 (en) * 1999-05-10 2000-11-16 Syngenta Participations Ag Regulation of viral gene expression
WO2003070972A2 (en) * 2002-02-20 2003-08-28 Sirna Therapeutics Inc. RNA INTERFERENCE MEDIATED INHIBITION OF CHROMOSOME TRANSLOCATION GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2003070918A2 (en) 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Rna interference by modified short interfering nucleic acid
WO2003070966A2 (en) * 2002-02-20 2003-08-28 Sirna Therapeutics, Inc RNA INTERFERENCE MEDIATED TARGET DISCOVERY AND TARGET VALIDATION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2003070914A2 (en) * 2002-02-20 2003-08-28 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF FOS GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2003076620A1 (en) 2002-03-14 2003-09-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for monitoring and modulating gene silencing
WO2004041838A1 (en) * 2002-11-01 2004-05-21 University Of Massachusetts Regulation of transcription elongation factors
EP1445321A1 (en) 2002-12-18 2004-08-11 Monsanto Technology LLC Maize embryo-specific promoter compositions and methods for use thereof
US6777588B2 (en) 2000-10-31 2004-08-17 Peter Waterhouse Methods and means for producing barley yellow dwarf virus resistant cereal plants
EP1516931A2 (en) 1998-05-26 2005-03-23 Syngenta Participations AG DSRNA-mediated regulation of gene expression in plants
WO2005028649A1 (en) 2002-02-20 2005-03-31 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
AU781598B2 (en) * 1999-04-21 2005-06-02 Alnylam Pharmaceuticals, Inc. Methods and compositions for inhibiting the function of polynucleotide sequences
EP1550719A1 (en) * 1999-01-30 2005-07-06 Alnylam Europe AG Methods and medicament for inhibition the expression of a defined gene
WO2005060739A1 (en) 2003-12-24 2005-07-07 G2 Inflammation Pty Ltd Transgenic non-human mammal comprising a polynucleotide encoding human or humanized c5ar
US6933146B2 (en) 2001-01-26 2005-08-23 Commonwealth Scientific And Industrial Research Corporation Methods and means for producing efficient silencing construct using recombinational cloning
WO2005077116A2 (en) 2004-02-10 2005-08-25 Mosanto Technology, Llc Recombinant dna for gene suppression
WO2005081714A2 (en) 2003-11-21 2005-09-09 Revivicor, Inc. Use of interfering rna in the production of transgenic animals
WO2005110068A2 (en) 2004-04-09 2005-11-24 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
EP1621632A1 (en) 2004-07-31 2006-02-01 Monsanto Technology, LLC Genes and uses for plant improvement
WO2005087926A3 (en) * 2004-03-05 2006-03-16 Benitec Inc Multiple promoter expression cassettes for simultaneous delivery of rnai agents
US7022828B2 (en) 2001-04-05 2006-04-04 Sirna Theraputics, Inc. siRNA treatment of diseases or conditions related to levels of IKK-gamma
WO2006046148A2 (en) 2004-10-25 2006-05-04 Devgen Nv Rna constructs
US7056704B2 (en) 2000-12-01 2006-06-06 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. RNA interference mediating small RNA molecules
US7067722B2 (en) 1999-08-26 2006-06-27 Monsanto Technology Llc Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
WO2003070193A3 (en) * 2002-02-20 2006-08-24 James Mcswiggen RNA interference mediated inhibition of HIV gene expression using short interfering nucleic acid (siNA)
WO2006099249A2 (en) 2005-03-10 2006-09-21 Monsanto Technology Llc Maize seed with synergistically enhanced lysine content
US7148336B2 (en) 1999-08-26 2006-12-12 Calgene Llc Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acid levels
WO2006133983A1 (en) 2005-04-19 2006-12-21 Basf Plant Science Gmbh Starchy-endosperm and/or germinating embryo-specific expression in mono-cotyledonous plants
US7166771B2 (en) 2002-06-21 2007-01-23 Monsanto Technology Llc Coordinated decrease and increase of gene expression of more than one gene using transgenic constructs
WO2007095469A2 (en) 2006-02-10 2007-08-23 Monsanto Technology Llc Identification and use of target genes for control of plant parasitic nematodes
WO2008116094A2 (en) 2007-03-21 2008-09-25 Brookhaven Science Associates, Llc Combined hairpin-antisense compositions and methods for modulating expression
US7452987B2 (en) 2002-08-05 2008-11-18 Silence Therapeutics Aktiengesellschaft (Ag) Interfering RNA molecules
EP2003205A2 (en) 2004-12-28 2008-12-17 Pioneer Hi-Bred International, Inc. Improved grain quality through altered expression of seed proteins
WO2008157263A2 (en) 2007-06-15 2008-12-24 Arkansas State University Methods of delivery of molecules to cells using a ricin subunit and compositions relating to same
US7473525B2 (en) 2001-01-09 2009-01-06 Alnylam Europe Ag Compositions and methods for inhibiting expression of anti-apoptotic genes
US7491805B2 (en) 2001-05-18 2009-02-17 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
WO2009021285A1 (en) 2007-08-13 2009-02-19 Commonwealth Scientific And Industrial Research Organisation Barley with low levels of hordein
WO2009026660A1 (en) 2007-08-30 2009-03-05 Walter And Eliza Hall Institute Of Medical Research Dendritic cell marker and uses thereof
US7531718B2 (en) 1999-08-26 2009-05-12 Monsanto Technology, L.L.C. Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
WO2009067580A2 (en) 2007-11-20 2009-05-28 Pioneer Hi-Bred International, Inc. Maize ethylene signaling genes and modulation of same for improved stress tolerance in plants
WO2009067751A1 (en) 2007-11-27 2009-06-04 Commonwealth Scientific And Industrial Research Organisation Plants with modified starch metabolism
US7560538B2 (en) 2003-11-05 2009-07-14 University Of Pittsburgh Porcine isogloboside 3 synthase protein, cDNA, genomic organization, and regulatory region
US7566813B2 (en) 2002-03-21 2009-07-28 Monsanto Technology, L.L.C. Nucleic acid constructs and methods for producing altered seed oil compositions
US7601888B2 (en) 2002-03-21 2009-10-13 Monsanto Technology L.L.C. Nucleic acid constructs and methods for producing altered seed oil compositions
WO2009129558A1 (en) 2008-04-24 2009-10-29 Newsouth Innovations Pty Limited Cyanobacteria saxitoxin gene cluster and detection of cyanotoxic organisms
US7612194B2 (en) 2001-07-24 2009-11-03 Monsanto Technology Llc Nucleic acid sequences from Diabrotica virgifera virgifera LeConte and uses thereof
EP2116607A1 (en) 2003-03-28 2009-11-11 Monsanto Technology, LLC Novel plant promoters for use in early seed development
WO2010005527A1 (en) 2008-06-30 2010-01-14 Angioblast Systems, Inc. Treatment of eye diseases and excessive neovascularization using a combined therapy
WO2010009499A1 (en) 2008-07-21 2010-01-28 Commonwealth Scientific And Industrial Research Organisation Improved cottonseed oil and uses
US7659390B2 (en) 2002-02-20 2010-02-09 Sirna Therapeutics, Inc. RNA interference mediated inhibition of muscarinic colinergic receptor gene expression using short interfering nucleic acid (siNA)
US7659389B2 (en) 2001-05-18 2010-02-09 Sirna Therapeutics, Inc. RNA interference mediated inhibition of MYC and/or MYB gene expression using short interfering nucleic acid (siNA)
US7662951B2 (en) 2000-08-30 2010-02-16 Sirna Therapeutics, Inc. RNA interference mediated treatment of Alzheimer's disease using short interfering nucleic acid (siNA)
US7662952B2 (en) 2002-02-20 2010-02-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of GRB2 associated binding protein (GAB2) gene expression using short interfering nucleic acid (siNA)
US7667030B2 (en) 2002-02-20 2010-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of matrix metalloproteinase 13 (MMP13) gene expression using short interfering nucleic acid (siNA)
US7667029B2 (en) 2002-02-20 2010-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of checkpoint kinase-1 (CHK-1) gene expression using short interfering nucleic acid (siNA)
US7678897B2 (en) 2002-02-20 2010-03-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)
US7691999B2 (en) 2002-02-20 2010-04-06 Sirna Therapeutics, Inc. RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering nucleic acid (siNA)
US7691995B2 (en) 2001-07-12 2010-04-06 University Of Massachusetts In vivo production of small interfering RNAS that mediate gene silencing
US7695426B2 (en) 2003-08-20 2010-04-13 Biological Resources Pty Ltd Methods for enhancing viability
US7700760B2 (en) 2002-02-20 2010-04-20 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular cell adhesion molecule (VCAM) gene expression using short interfering nucleic acid (siNA)
US7732417B2 (en) 2000-03-16 2010-06-08 Cold Spring Harbor Laboratory Methods and compositions for RNA interference using recombinant Dicer and Argonaut
WO2010065867A1 (en) 2008-12-04 2010-06-10 Pioneer Hi-Bred International, Inc. Methods and compositions for enhanced yield by targeted expression of knotted1
US20100145015A1 (en) * 2007-03-26 2010-06-10 Uri Galili Compositions and methods for increasing immunogenicity of glycoprotein vaccines
WO2010066689A2 (en) 2008-12-09 2010-06-17 Novartis Ag Organic compounds
US7767802B2 (en) 2001-01-09 2010-08-03 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
WO2010101818A1 (en) 2009-03-02 2010-09-10 Pioneer Hi-Bred International, Inc. Nac transcriptional activators involved in abiotic stress tolerance
US7795493B2 (en) 2002-08-21 2010-09-14 Revivicor, Inc. Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US7795422B2 (en) 2002-02-20 2010-09-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hypoxia inducible factor 1 (HIF1) gene expression using short interfering nucleic acid (siNA)
WO2010118477A1 (en) 2009-04-17 2010-10-21 Molecular Plant Breeding Nominees Ltd Plant promoter operable in endosperm and uses thereof
WO2010120862A1 (en) 2009-04-14 2010-10-21 Pioneer Hi-Bred International, Inc. Modulation of acc synthase improves plant yield under low nitrogen conditions
WO2010123904A1 (en) 2009-04-20 2010-10-28 Monsanto Technology Llc Multiple virus resistance in plants
US7829693B2 (en) 1999-11-24 2010-11-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
US7833992B2 (en) 2001-05-18 2010-11-16 Merck Sharpe & Dohme Conjugates and compositions for cellular delivery
EP2251349A1 (en) 2006-04-19 2010-11-17 Pioneer Hi-Bred International, Inc. Isolated polynucleotide molecules corresponding to mutant and wild-type alleles of the maize D9 gene and methods of use
WO2010139026A1 (en) 2009-06-05 2010-12-09 Centenary Institute Of Cancer Medicine And Cell Biology Therapeutic and diagnostic molecules
EP2261362A2 (en) 2005-05-25 2010-12-15 Pioneer Hi-Bred International Inc. Methods for improving crop plant architecture and yield
US7855323B2 (en) 2004-02-10 2010-12-21 Monsanto Technology Llc Recombinant DNA for gene suppression
US7858625B2 (en) 2001-05-18 2010-12-28 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US7858769B2 (en) 2004-02-10 2010-12-28 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using multifunctional short interfering nucleic acid (multifunctional siNA)
US7868160B2 (en) 2001-01-09 2011-01-11 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
EP2275563A2 (en) 2005-09-16 2011-01-19 deVGen N.V. Transgenic plant-based methods for plant insect pests using RNAi
WO2011011273A1 (en) 2009-07-24 2011-01-27 Pioneer Hi-Bred International, Inc. The use of dimerization domain component stacks to modulate plant architecture
US7884264B2 (en) 2006-01-17 2011-02-08 Biolex Therapeutics, Inc. Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in duckweed plants
US7893248B2 (en) 2002-02-20 2011-02-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Myc and/or Myb gene expression using short interfering nucleic acid (siNA)
US7897752B2 (en) 2002-02-20 2011-03-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of telomerase gene expression using short interfering nucleic acid (siNA)
US7897753B2 (en) 2002-02-20 2011-03-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of XIAP gene expression using short interfering nucleic acid (siNA)
US7897757B2 (en) 2002-02-20 2011-03-01 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of protein tyrosine phosphatase-1B (PTP-1B) gene expression using short interfering nucleic acid (siNA)
EP2292739A1 (en) 2006-03-24 2011-03-09 Institut National De La Recherche Agronomique Method for preparing differentiated avian cells and genes involved in the maintenance of pluripotency
EP2292773A1 (en) 2004-03-25 2011-03-09 Monsanto Technology LLC Genes and uses for plant improvement
US7910724B2 (en) 2002-02-20 2011-03-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Fos gene expression using short interfering nucleic acid (siNA)
US7910725B2 (en) 2002-02-20 2011-03-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (siNA)
US7915400B2 (en) 2002-02-20 2011-03-29 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of hepatitis C virus (HCV) gene expression using short interfering nucleic acid (siNA)
WO2011041796A1 (en) 2009-10-02 2011-04-07 Pioneer Hi-Bred International, Inc. Down-regulation of acc synthase for improved plant performance
US7923547B2 (en) 2002-09-05 2011-04-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US7923549B2 (en) 2002-02-20 2011-04-12 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (siNA)
US7928219B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of placental growth factor gene expression using short interfering nucleic acid (SINA)
US7928220B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of stromal cell-derived factor-1 (SDF-1) gene expression using short interfering nucleic acid (siNA)
US7928218B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of polycomb group protein EZH2 gene expression using short interfering nucleic acid (siNA)
US7935812B2 (en) 2002-02-20 2011-05-03 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of hepatitis C virus (HCV) expression using short interfering nucleic acid (siNA)
US7943819B2 (en) 2005-09-16 2011-05-17 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
US7943757B2 (en) 2002-02-20 2011-05-17 Mcswiggen James RNA interference mediated inhibition of intercellular adhesion molecule (ICAM) gene expression using short interfering nucleic acid (siNA)
EP2267139A3 (en) * 1998-04-08 2011-05-25 Commonwealth Scientific and Industrial Research Organization Methods and means for obtaining modified phenotypes
US7960614B2 (en) 2003-06-06 2011-06-14 Arborgen, Llc Plant transformation and selection
EP2333061A1 (en) 2001-07-06 2011-06-15 Commonwealth Scientific and Industrial Research Organization Delivery of dsRNA to arthropods
WO2011073326A2 (en) 2009-12-18 2011-06-23 Novartis Ag Organic compositions to treat hsf1-related diseases
EP2338905A2 (en) 2005-02-23 2011-06-29 North Carolina State University Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes
US7977472B2 (en) 2002-02-20 2011-07-12 Leonid Beigelman RNA interference mediated inhibition of myostatin gene expression using short interfering nucleic acid (siNA)
WO2011085062A1 (en) 2010-01-06 2011-07-14 Pioneer Hi-Bred International, Inc. Identification of diurnal rhythms in photosynthetic and non-photosynthetic tissues from zea mays and use in improving crop plants
US7985853B2 (en) 2002-02-20 2011-07-26 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of platelet derived growth factor (PDGF) and platelet derived growth factor receptor (PDGFR) gene expression using short interfering nucleic acid (siNA)
US7989612B2 (en) 2002-02-20 2011-08-02 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
EP2357244A2 (en) 2004-04-22 2011-08-17 Commonwealth Scientific and Industrial Research Organisation Synthesis of long-chain polyunsaturated fatty acids by recombinant cells.
WO2011098449A1 (en) 2010-02-10 2011-08-18 Novartis Ag Methods and compounds for muscle growth
US8008473B2 (en) 2002-02-20 2011-08-30 Mcswiggen James RNA interference mediated inhibition of TNF and TNF receptor gene expression using short interfering nucleic acid (siNA)
US8008472B2 (en) 2001-05-29 2011-08-30 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of human immunodeficiency virus (HIV) gene expression using short interfering nucleic acid (siNA)
US8013143B2 (en) 2002-02-20 2011-09-06 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of CXCR4 gene expression using short interfering nucleic acid (siNA)
US8017761B2 (en) 2001-05-18 2011-09-13 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of Stearoyl-CoA desaturase (SCD) gene expression using short interfering nucelic acid (siNA)
US8049069B2 (en) 2004-03-31 2011-11-01 Commonwealth Scientific And Industrial Research Organisation Genes involved in plant fibre development
US8067575B2 (en) 2002-02-20 2011-11-29 Merck, Sharp & Dohme Corp. RNA interference mediated inhibition of cyclin D1 gene expression using short interfering nucleic acid (siNA)
US8093369B2 (en) 2005-10-11 2012-01-10 Ben Gurion University Of The Negev Research And Development Authority Ltd. Compositions for silencing the expression of VDAC1 and uses thereof
US8093043B2 (en) 2008-06-04 2012-01-10 New York University β-TrCP1, β-TrCP2 and RSK1 or RSK2 inhibitors and methods for sensitizing target cells to apoptosis
WO2012007945A2 (en) 2010-07-12 2012-01-19 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization, (A.R.O.), Volcani Center Isolated polynucleotides and methods and plants using same for regulating plant acidity
US8106251B2 (en) 2002-08-21 2012-01-31 Revivicor, Inc. Tissue products derived from porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase
EP2426206A2 (en) 2006-02-13 2012-03-07 Monsanto Technology LLC Selecting and stablizing dsRNA constructs
US8153776B2 (en) 2000-03-16 2012-04-10 Cold Spring Harbor Laboratory Methods and compositions for RNA interference
WO2012055362A1 (en) 2010-10-28 2012-05-03 Benitec Biopharma Limited Hbv treatment
WO2012058726A1 (en) 2010-11-05 2012-05-10 Transbio Ltd Markers of endothelial progenitor cells and uses thereof
WO2012058730A1 (en) 2010-11-04 2012-05-10 Arista Cereal Technologies Pty Ltd High amylose wheat
JP2012085641A (en) * 1999-11-19 2012-05-10 Cancer Research Technology Ltd Inhibition of gene expression with dsrna
EP2489726A2 (en) 2005-01-12 2012-08-22 Monsanto Technology LLC Genes and uses for plant improvement
WO2012112586A1 (en) 2011-02-14 2012-08-23 Revivicor, Inc. Genetically modified pigs for xenotransplantation of vascularized xenografts and derivatives thereof
US8258288B2 (en) 2002-02-20 2012-09-04 Sirna Therapeutics, Inc. RNA interference mediated inhibition of respiratory syncytial virus (RSV) expression using short interfering nucleic acid (siNA)
US8269082B2 (en) 2005-10-20 2012-09-18 Commonwealth Scientific And Industrial Research Organisation Cereals with altered dormancy
US8273866B2 (en) 2002-02-20 2012-09-25 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
WO2012129373A2 (en) 2011-03-23 2012-09-27 Pioneer Hi-Bred International, Inc. Methods for producing a complex transgenic trait locus
US8299042B2 (en) 2002-04-26 2012-10-30 Alnylam Pharmaceuticals, Inc. Methods and compositions for silencing genes without inducing toxicity
WO2012148835A1 (en) 2011-04-29 2012-11-01 Pioneer Hi-Bred International, Inc. Down-regulation of a homeodomain-leucine zipper i-class homeobox gene for improved plant performance
US8329890B2 (en) 2002-08-05 2012-12-11 University Of Iowa Research Foundation SiRNA-mediated gene silencing
US8329667B2 (en) 2004-04-23 2012-12-11 The Trustees Of Columbia University In The City Of New York Inhibition of hairless protein mRNA
US8329989B2 (en) 2008-09-29 2012-12-11 Monsanto Technology Llc Soybean transgenic event MON87705 and methods for detection thereof
WO2012174139A2 (en) 2011-06-14 2012-12-20 Synthon Biopharmaceuticals B.V. Compositions and methods for making and b ioc ont aining auxotrophic transgenic plants
US8394628B2 (en) 2000-03-30 2013-03-12 University Of Massachusetts RNA sequence-specific mediators of RNA interference
WO2013066805A1 (en) 2011-10-31 2013-05-10 Pioneer Hi-Bred International, Inc. Improving plant drought tolerance, nitrogen use efficiency and yield
WO2013063653A1 (en) 2011-11-04 2013-05-10 Arista Cereal Technologies Pty Limited High amylose wheat - ii
WO2013066423A2 (en) 2011-06-21 2013-05-10 Pioneer Hi-Bred International, Inc. Methods and compositions for producing male sterile plants
US8461418B2 (en) 2004-08-11 2013-06-11 Monsanto Technology Llc Enhanced zein reduction in transgenic corn seed
WO2013088438A1 (en) 2011-12-11 2013-06-20 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization, (A.R.O.), Volcani Center Methods of modulating stomata conductance and plant expression constructs for executing same
WO2013096991A1 (en) 2011-12-27 2013-07-04 Commonwealth Scientific And Industrial Research Organisation Production of dihydrosterculic acid and derivatives thereof
WO2013096992A1 (en) 2011-12-27 2013-07-04 Commonwealth Scientific And Industrial Reserach Organisation Simultaneous gene silencing and supressing gene silencing in the same cell
WO2013096993A1 (en) 2011-12-27 2013-07-04 Commonwealth Scientific And Industrial Research Organisation Processes for producing lipids
US8481710B2 (en) 2002-08-05 2013-07-09 University Of Iowa Research Foundation RNA interference suppression of neurodegenerative diseases and methods of use thereof
WO2013105022A2 (en) 2012-01-09 2013-07-18 Novartis Ag Organic compositions to treat beta-catenin-related diseases
WO2013104026A1 (en) 2012-01-11 2013-07-18 The Australian National University Method for modulating plant root architecture
US8524879B2 (en) 2002-08-05 2013-09-03 University Of Iowa Research Foundation RNA interference suppresion of neurodegenerative diseases and methods of use thereof
US8536429B2 (en) 2006-07-12 2013-09-17 Commonwealth Scientific And Industrial Research Organisation Polynucleotides encoding a NAX2 polypeptide and methods for enhancing salinity tolerance in plants
WO2013138358A1 (en) 2012-03-13 2013-09-19 Pioneer Hi-Bred International, Inc. Genetic reduction of male fertility in plants
WO2013138309A1 (en) 2012-03-13 2013-09-19 Pioneer Hi-Bred International, Inc. Genetic reduction of male fertility in plants
WO2013159149A1 (en) 2012-04-25 2013-10-31 Commonwealth Scientific And Industrial Research Organisation High oleic acid oils
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
US8633028B2 (en) * 2003-07-02 2014-01-21 Musc Foundation For Research Development dsRNA induced specific and non-specific immunity in crustaceans and other invertebrates and biodelivery vehicles for use therein
WO2014027021A1 (en) 2012-08-16 2014-02-20 Vib Vzw Means and methods for altering the lignin pathway in plants
US8716554B2 (en) 2003-08-21 2014-05-06 Rahan Meristem (1998) Ltd. Plant Propagation & Biotechnology Plants resistant to cytoplasm-feeding parasites
EP2730587A2 (en) 2006-02-09 2014-05-14 Pioneer Hi-Bred International, Inc. Genes for enhancing nitrogen utilization efficiency in crop plants
WO2014118123A1 (en) 2013-01-29 2014-08-07 The University Court Of The University Of Glasgow Methods and means for increasing stress tolerance and biomass in plants
WO2014143996A2 (en) 2013-03-15 2014-09-18 Pioneer Hi-Bred International, Inc. Compositions and methods of use of acc oxidase polynucleotides and polypeptides
WO2014147249A1 (en) 2013-03-21 2014-09-25 Vib Vzw Means and methods for the reduction of photorespiration in crops
WO2014160122A1 (en) 2013-03-14 2014-10-02 Pioneer Hi-Bred International, Inc. Maize stress related transcription factor 18 and uses thereof
WO2014164116A1 (en) 2013-03-13 2014-10-09 Pioneer Hi-Bred International, Inc. Functional expression of bacterial major facilitator superfamily (sfm) gene in maize to improve agronomic traits and grain yield
WO2014164014A1 (en) 2013-03-11 2014-10-09 Pioneer Hi-Bred International, Inc. Genes for improving nutrient uptake and abiotic stress tolerance in plants
WO2014164074A1 (en) 2013-03-13 2014-10-09 Pioneer Hi-Bred International, Inc. Enhanced nitrate uptake and nitrate translocation by over-expressing maize functional low-affinity nitrate transporters in transgenic maize
US8895805B2 (en) 2006-12-04 2014-11-25 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences Method for modifying insect resistance of plants by utilizing RNAi technique
EP2810952A1 (en) 2013-06-03 2014-12-10 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Novel pest control methods
WO2014200842A2 (en) 2013-06-11 2014-12-18 Syngenta Participations Ag Methods for generating transgenic plants
WO2014205516A1 (en) 2013-06-25 2014-12-31 The Walter And Eliza Hall Institute Of Medical Research Method of treating intracellular infection
EP2821490A2 (en) 2008-10-30 2015-01-07 Pioneer Hi-Bred International Inc. Manipulation of glutamine synthetases (GS) to improve nitrogen use efficiency and grain yield in higher plants
US8945884B2 (en) 2000-12-11 2015-02-03 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites
US9018179B2 (en) 2001-07-23 2015-04-28 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals
US9029527B2 (en) 1998-03-20 2015-05-12 Commonwealth Scientific And Industrial Research Organisation Synthetic genes and genetic constructs
WO2015069459A1 (en) 2013-11-05 2015-05-14 Novartis Ag Organic compounds
US9051566B2 (en) 2001-01-31 2015-06-09 Alnylam Pharmaceuticals, Inc. Post-transcriptional gene silencing using expressed double stranded RNA
WO2015089585A1 (en) 2013-12-18 2015-06-25 Csl Limited Method of treating wounds
US9074213B2 (en) 2001-01-09 2015-07-07 Alnylam Pharmacuticals, Inc. Compositions and methods for inhibiting expression of a target gene
WO2015116680A1 (en) 2014-01-30 2015-08-06 Two Blades Foundation Plants with enhanced resistance to phytophthora
US9102939B2 (en) 1997-12-23 2015-08-11 The Carnegie Institution Of Washington Genetic inhibition by double-stranded RNA
US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
WO2015171603A1 (en) 2014-05-06 2015-11-12 Two Blades Foundation Methods for producing plants with enhanced resistance to oomycete pathogens
EP2966157A1 (en) 2014-07-07 2016-01-13 Commonwealth Scientific and Industrial Research Organisation Processes for producing industrial products from plant lipids
WO2016005449A1 (en) 2014-07-08 2016-01-14 Vib Vzw Means and methods to increase plant yield
EP2980220A1 (en) 2005-09-20 2016-02-03 BASF Plant Science GmbH Improved methods controlling gene expression
US9260471B2 (en) 2010-10-29 2016-02-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
US9267145B2 (en) 2002-05-03 2016-02-23 Duke University Method of regulating gene expression
US9290777B2 (en) 2007-02-05 2016-03-22 National University Of Singapore Putative cytokinin receptor and methods for use thereof
US9309512B2 (en) 2006-08-31 2016-04-12 Monsanto Technology Llc Phased small RNAs
WO2016079527A1 (en) 2014-11-19 2016-05-26 Tetralogic Birinapant Uk Ltd Combination therapy
WO2016097773A1 (en) 2014-12-19 2016-06-23 Children's Cancer Institute Therapeutic iap antagonists for treating proliferative disorders
US9441239B2 (en) 1998-04-08 2016-09-13 Commonwealth Scientific & Industrial Research Organisation Methods and means for obtaining modified phenotypes
WO2016196489A1 (en) 2015-05-29 2016-12-08 Arcadia Biosciences Reduced gluten grains and compositions thereof
US9534252B2 (en) 2003-12-01 2017-01-03 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
US9592250B2 (en) 2002-02-01 2017-03-14 Life Technologies Corporation Double-stranded oligonucleotides
WO2017062790A1 (en) 2015-10-09 2017-04-13 Two Blades Foundation Cold shock protein receptors and methods of use
US9657294B2 (en) 2002-02-20 2017-05-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
WO2017083920A1 (en) 2015-11-18 2017-05-26 Commonwealth Scientific And Industrial Research Organisation Rice grain with thickened aleurone
WO2017091952A1 (en) 2015-11-30 2017-06-08 谢彦晖 Use of akt2 in diagnosis and treatment of tumor
US9708621B2 (en) 1999-08-13 2017-07-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US9765351B2 (en) 2006-02-13 2017-09-19 Monsanto Technology Llc Modified gene silencing
WO2017161264A1 (en) 2016-03-18 2017-09-21 Pioneer Hi-Bred International, Inc. Methods and compositions for producing clonal, non-reduced, non-recombined gametes
US9777275B2 (en) 2002-02-01 2017-10-03 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US9803008B2 (en) 2013-11-28 2017-10-31 Csl Limited Method of treating diabetic nephropathy by administering antibodies to vascular endothelial growth factor B (VEGF-B)
US9885038B2 (en) 2007-08-14 2018-02-06 Commonwealth Scientific & Industrial Research Organisation Gene silencing methods
EP3293257A1 (en) 2009-03-20 2018-03-14 Mesoblast, Inc. Production of reprogrammed pluripotent cells
CN107841510A (en) * 2016-09-20 2018-03-27 中国科学院青岛生物能源与过程研究所 A kind of method of prokaryotic post-transcriptional level control different genes expression ratio
US9963698B2 (en) 1998-03-20 2018-05-08 Commonwealth Scientific And Industrial Research Organisation Control of gene expression
US9994853B2 (en) 2001-05-18 2018-06-12 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
US10428336B2 (en) 2013-10-16 2019-10-01 The Australian National University Method for modulating plant growth
US10508277B2 (en) 2004-05-24 2019-12-17 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
US10517887B2 (en) 2001-07-23 2019-12-31 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals
WO2020078865A1 (en) 2018-10-16 2020-04-23 F. Hoffmann-La Roche Ag Use of akt inhibitors in ophthalmology
WO2020148310A1 (en) 2019-01-17 2020-07-23 F. Hoffmann-La Roche Ag E3 ubiquitin ligase (ube3a) protein targets
US10849345B2 (en) 2013-06-13 2020-12-01 Commonwealth Scientific And Industrial Research Organisation Barley with very low levels of hordeins
WO2021019536A1 (en) 2019-07-30 2021-02-04 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Methods of controlling cannabinoid synthesis in plants or cells and plants and cells produced thereby
EP3825408A1 (en) 2019-11-19 2021-05-26 FRAUNHOFER-GESELLSCHAFT zur Förderung der angewandten Forschung e.V. Methods of multi-species insect pest control
WO2022162015A1 (en) 2021-01-26 2022-08-04 Universite Brest Bretagne Occidentale Novel stim1 splicing variants and uses thereof
WO2023012342A1 (en) 2021-08-06 2023-02-09 Kws Vegetables B.V. Durable downy mildew resistance in spinach
WO2023020980A1 (en) 2021-08-16 2023-02-23 F. Hoffmann-La Roche Ag E3 ubiquitin ligase (ube3a) protein targets
US11932854B2 (en) 2021-10-25 2024-03-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)

Families Citing this family (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6939712B1 (en) * 1998-12-29 2005-09-06 Impedagen, Llc Muting gene activity using a transgenic nucleic acid
CA2361201A1 (en) * 1999-01-28 2000-08-03 Medical College Of Georgia Research Institute, Inc. Composition and method for in vivo and in vitro attenuation of gene expression using double stranded rna
AU2008202208C1 (en) * 1999-01-30 2014-04-24 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a defined gene
US20040138168A1 (en) * 1999-04-21 2004-07-15 Wyeth Methods and compositions for inhibiting the function of polynucleotide sequences
CN1426466A (en) * 2000-03-17 2003-06-25 贝尼泰克澳大利亚有限公司 Genetic silencing
US20050037989A1 (en) * 2001-08-27 2005-02-17 Lewis David L. Inhibition of gene function by delivery of polynucleotide-based gene expression inhibitors to mammalian cells in vivo
WO2004015062A2 (en) 2002-08-12 2004-02-19 New England Biolabs, Inc. Methods and compositions relating to gene silencing
US20040242518A1 (en) * 2002-09-28 2004-12-02 Massachusetts Institute Of Technology Influenza therapeutic
JP2006526394A (en) * 2003-06-03 2006-11-24 ベニテック オーストラリア リミテッド Double-stranded nucleic acid
EP1636385A4 (en) * 2003-06-24 2010-06-02 Mirus Bio Corp Inhibition of gene function by delivery of polynucleotide-based gene expression inhibitors to mammalian cells in vivo
WO2005009134A1 (en) * 2003-07-21 2005-02-03 Lifecell Corporation ACELLULAR TISSUE MATRICES MADE FROM GALACTOSE α-1,3-GALACTOSE-DEFICIENT TISSUE
US20080044906A1 (en) * 2003-09-12 2008-02-21 Peter Michael Waterhouse Modified Gene-Silencing Nucleic Acid Molecules and Uses Thereof
MX2007004310A (en) 2004-10-13 2007-06-18 Univ Georgia Res Found Nematode resistant transgenic plants.
US7368086B2 (en) * 2004-10-29 2008-05-06 Invitrogen Corporation Functionalized fluorescent nanocrystals, and methods for their preparation and use
US8404927B2 (en) * 2004-12-21 2013-03-26 Monsanto Technology Llc Double-stranded RNA stabilized in planta
DE202005004135U1 (en) * 2005-03-11 2005-05-19 Klocke Verpackungs-Service Gmbh Multi-component packaging with applicator
CA2605068A1 (en) * 2005-04-15 2006-10-26 The Board Of Regents Of The University Of Texas System Delivery of sirna by neutral lipid compositions
WO2006113743A2 (en) * 2005-04-18 2006-10-26 Massachusetts Institute Of Technology Compositions and methods for rna interference with sialidase expression and uses thereof
WO2008086325A1 (en) 2007-01-10 2008-07-17 Sanofi-Aventis Method for determining the stability of organic methyleneamines in the presence of semicarbazide-sensitive amine oxidase
US8067390B2 (en) * 2007-03-02 2011-11-29 The Board Of Regents Of The University Of Texas System Therapeutic targeting of interleukins using siRNA in neutral liposomes
US8809626B2 (en) 2007-12-21 2014-08-19 Keygene N.V. Trichome specific promoters
CN102056605A (en) * 2008-04-04 2011-05-11 罗伯特·绍尔 Pharmaceutical composition
WO2009132351A2 (en) * 2008-04-25 2009-10-29 University Of Medicine And Dentistry Of New Jersey Anti-sense microrna expression vectors
CN102272157B (en) 2008-11-07 2015-11-25 研究发展基金会 For suppressing composition and the method for the formation of CRIPTO/GRP78 mixture and signal
US8734853B2 (en) 2008-11-17 2014-05-27 University Of North Texas Health Science Center At Fort Worth HDL particles for delivery of nucleic acids
US20110287088A1 (en) 2008-12-03 2011-11-24 Research Development Foundation Modulation of olfml-3 mediated angiogenesis
US20110045080A1 (en) * 2009-03-24 2011-02-24 William Marsh Rice University Single-Walled Carbon Nanotube/Bioactive Substance Complexes and Methods Related Thereto
WO2010142465A1 (en) 2009-06-08 2010-12-16 Nunhems B.V. Drought tolerant plants
CN101760555B (en) * 2009-09-30 2012-06-27 西南大学 Method for identifying single-copy transgenic tobacco based on PCR technology
CN107090453A (en) * 2010-05-10 2017-08-25 德克萨斯A&M大学系统 The composition of expressing gene product, organism, system and method in plant
CN107974457A (en) 2010-05-28 2018-05-01 纽海姆有限公司 The plant of fruit size increase
EP3192880B1 (en) 2010-08-18 2019-10-09 Fred Hutchinson Cancer Research Center Nucleic acid agents for use in treating facioscapulohumeral dystrophy (fshd)
US9233997B2 (en) * 2010-08-26 2016-01-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of prolyl hydroxylase domain 2 (PHD2) gene expression using short interfering nucleic acid (siNA)
US20120107355A1 (en) 2010-10-27 2012-05-03 Harrisvaccines, Inc. Method of rapidly producing improved vaccines for animals
CN102719454B (en) * 2012-06-15 2014-07-30 华南农业大学 Optimized sulfide quinine oxidation-reduction enzyme gene and expression vector thereof
WO2014045126A2 (en) 2012-09-18 2014-03-27 Uti Limited Partnership Treatment of pain by inhibition of usp5 de-ubiquitinase
CN105143470B (en) 2013-02-28 2020-06-09 德克萨斯大学系统董事会 Methods for classifying cancer as susceptible to TMEPAI-directed therapy and treating the cancer
ES2846758T3 (en) 2013-11-04 2021-07-29 Lifecell Corp Methods to remove alpha-galactose
CA2933590C (en) 2013-12-11 2023-10-03 Sloan-Kettering Institute For Cancer Research Glucocorticoid inhibitors for treatment of prostate cancer
WO2015171723A1 (en) 2014-05-06 2015-11-12 Research Development Foundation Methods for treating insulin resistance and for sensitizing patients to glp1 agonist therapy
US9517270B2 (en) 2014-12-08 2016-12-13 The Board Of Regents Of The University Of Texas System Lipocationic polymers and uses thereof
US10000766B2 (en) * 2015-07-17 2018-06-19 National Chung Hsing University Recombinant construct, recombinant microorganism, recombinant plant cell and method of providing plant with resistance against DNA virus and RNA virus
JP6978152B2 (en) 2015-09-04 2021-12-08 キージーン ナムローゼ フェンノートシャップ Multiphase spore reproductive gene
EP3349802B1 (en) 2015-09-14 2021-08-04 The Board of Regents of the University of Texas System Lipocationic dendrimers and uses thereof
WO2017162265A1 (en) * 2016-03-21 2017-09-28 Biontech Rna Pharmaceuticals Gmbh Trans-replicating rna
AU2017267634C1 (en) 2016-05-16 2022-05-26 The Board Of Regents Of The University Of Texas System Cationic sulfonamide amino lipids and amphiphilic zwitterionic amino lipids
WO2018035451A1 (en) 2016-08-19 2018-02-22 Calimmune, Inc. Methods and compositions for treating conditions using recombinant self-complementary adeno-associated virus
EP3500279A4 (en) * 2016-08-19 2020-04-22 Calimmune, Inc. Methods and compositions for treating canine conditions using recombinant self-complementary adeno-associated virus
JP2021519587A (en) 2018-03-30 2021-08-12 ユニバーシティ オブ ジュネーブ MicroRNA expression constructs and their use
EP3920889A4 (en) 2019-02-08 2022-12-07 Board of Regents, The University of Texas System Telomerase-containing exosomes for treatment of diseases associated with aging and age-related organ dysfunction
AU2021260578A1 (en) 2020-04-20 2022-11-03 Board Of Regents, The University Of Texas System Biologically active dry powder compositions and method of their manufacture and use
IL298558A (en) 2020-05-27 2023-01-01 Antion Biosciences Sa Adapter molecules to re-direct car t cells to an antigen of interest
US20230266325A1 (en) 2020-06-30 2023-08-24 Lunglife Ai, Inc. Methods for detecting lung cancer
GB202206507D0 (en) 2022-05-04 2022-06-15 Antion Biosciences Sa Expression construct
WO2023230531A1 (en) 2022-05-24 2023-11-30 Lunglife Ai, Inc. Methods for detecting circulating genetically abnormal cells
WO2024028794A1 (en) 2022-08-02 2024-02-08 Temple Therapeutics BV Methods for treating endometrial and ovarian hyperproliferative disorders

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998053083A1 (en) * 1997-05-21 1998-11-26 Zeneca Limited Gene silencing

Family Cites Families (228)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US27783A (en) * 1860-04-10 Improvement in apparatus for evaporating sugar-juices
US114784A (en) * 1871-05-16 Improvement in compounds for cure of chills and fever
US86356A (en) * 1869-02-02 Improvement in the construction of fire-proof safes
US266005A (en) * 1882-10-17 William allen
US165894A (en) * 1875-07-20 Improvement in vessels for removing foul water from docks
US74684A (en) * 1868-02-18 of wallingford
US36197A (en) * 1862-08-12 Improvement in compound bullets for small-arms
US56235A (en) * 1866-07-10 Improvement in paring-knives
US3931397A (en) 1971-11-05 1976-01-06 Beecham Group Limited Biologically active material
US4024222A (en) 1973-10-30 1977-05-17 The Johns Hopkins University Nucleic acid complexes
US4034323A (en) * 1975-03-24 1977-07-05 Oki Electric Industry Company, Ltd. Magnetic relay
US4283393A (en) 1979-03-13 1981-08-11 Merck & Co., Inc. Topical application of interferon inducers
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US4605394A (en) 1982-12-03 1986-08-12 Simon V. Skurkovich Methods for the treatment of pathological conditions by removing interferon from the organism
DE3208247A1 (en) 1982-03-08 1983-09-22 Philips Patentverwaltung Gmbh, 2000 Hamburg CUVETTE FOR THE ATOMIC ABSORPTION SPECTROMETRY
JPS5936698A (en) 1982-08-20 1984-02-28 Science & Tech Agency Recombinant dna recombined with gene of virus of hepatitis b, transformed animal cell, and preparation of protein of virus of hepatitis b
NZ209840A (en) * 1983-10-17 1988-11-29 Kaji Akira A method of inhibiting viral propagation by hybridising dna with the viral rna thus blocking its action
US5190931A (en) * 1983-10-20 1993-03-02 The Research Foundation Of State University Of New York Regulation of gene expression by employing translational inhibition of MRNA utilizing interfering complementary MRNA
ATE198350T1 (en) 1983-10-20 2001-01-15 Univ New York State Res Found REGULATION OF GENE EXPRESSION THROUGH TRANSLATION INHIBITION USING A M-RNS DISABLED COMPLEMENTARY RNA
US5272065A (en) * 1983-10-20 1993-12-21 Research Foundation Of State University Of New York Regulation of gene expression by employing translational inhibition of MRNA utilizing interfering complementary MRNA
US5208149A (en) * 1983-10-20 1993-05-04 The Research Foundation Of State University Of New York Nucleic acid constructs containing stable stem and loop structures
US5173410A (en) 1984-02-15 1992-12-22 Lubrizol Genetics Inc. Transfer vector
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US4766072A (en) 1985-07-17 1988-08-23 Promega Corporation Vectors for in vitro production of RNA copies of either strand of a cloned DNA sequence
GB8521646D0 (en) 1985-08-30 1985-10-02 English Clays Lovering Pochin Inorganic fillers
CA1326450C (en) 1985-08-26 1994-01-25 William A. Carter Modulation of aids virus-related events by double stranded rnas (dsrnas)
US6617496B1 (en) 1985-10-16 2003-09-09 Monsanto Company Effecting virus resistance in plants through the use of negative strand RNAs
GB8601680D0 (en) 1986-01-23 1986-02-26 Agricultural Genetics Co Modification of plant viruses
IL81737A (en) 1986-03-28 1992-11-15 Calgene Inc Regulation of gene expression in plant cells
US5453566A (en) 1986-03-28 1995-09-26 Calgene, Inc. Antisense regulation of gene expression in plant/cells
US5107065A (en) 1986-03-28 1992-04-21 Calgene, Inc. Anti-sense regulation of gene expression in plant cells
US5017488A (en) * 1986-04-01 1991-05-21 University Of Medicine And Dentistry Of New Jersey Highly efficient dual T7/T3 promoter vector PJKF16 and dual SP6/T3 promoter vector PJFK15
EP0278667B1 (en) * 1987-02-09 1994-07-20 Mycogen Plant Science, Inc. Hybrid RNA virus
PH24467A (en) 1987-03-03 1990-07-18 Hem Res Inc Synergistics interplay of lymphokines and dsrnas
US4950652A (en) 1987-03-23 1990-08-21 Hem Research, Inc. dsRNAs for combination therapy in the treatment of viral diseases
AU1820588A (en) 1987-07-17 1989-01-19 Hem Research, Inc. Double-stranded rna correction of abnormalities in circulating immune complexes and monocyte function
IE66830B1 (en) 1987-08-12 1996-02-07 Hem Res Inc Topically active compositions of double-stranded RNAs
IE72103B1 (en) 1987-08-12 1997-03-12 Hem Res Inc Promotion of host defense by systemic dsRNA treatment
EP0306347B1 (en) 1987-09-04 1995-05-10 Hem Pharmaceuticals Corp. Diagnosis of double-stranded RNA deficiency states
US5874555A (en) 1987-10-30 1999-02-23 California Institute Of Technology Triple helices and processes for making same
US4963532A (en) 1987-11-25 1990-10-16 Hem Research, Inc. dsRNA-based prevention of viral escape
JP3046318B2 (en) 1987-12-15 2000-05-29 ジーン・シアーズ・ピーティーワイ・リミテッド Ribozyme
US5254678A (en) * 1987-12-15 1993-10-19 Gene Shears Pty. Limited Ribozymes
PH25365A (en) 1987-12-23 1991-05-13 Hem Research Rhase l inhibitor as a marker for virus infections
US5922602A (en) 1988-02-26 1999-07-13 Biosource Technologies, Inc. Cytoplasmic inhibition of gene expression
GB8810120D0 (en) 1988-04-28 1988-06-02 Plant Genetic Systems Nv Transgenic nuclear male sterile plants
CA1320446C (en) 1988-06-20 1993-07-20 William A. Carter Modulation of lymphokine-resistant cellular states by dsrnas
ATE103493T1 (en) 1988-07-07 1994-04-15 Hem Pharma Corp DIAGNOSIS AND TREATMENT OF CHRONIC FATIGUE.
US5597718A (en) * 1988-10-04 1997-01-28 Agracetus Genetically engineering cotton plants for altered fiber
US5198346A (en) * 1989-01-06 1993-03-30 Protein Engineering Corp. Generation and selection of novel DNA-binding proteins and polypeptides
US5434070A (en) * 1989-02-24 1995-07-18 The University Of Medicine And Dentistry Of New Jersey Reverse transcriptases from Escherichia coli and Myxococcus xanthus
US5780269A (en) 1989-02-24 1998-07-14 The University Of Medicine And Denistry Of New Jersey Hybrid molecules
US5405775A (en) * 1989-02-24 1995-04-11 The University Of Medicine And Dentistry Of New Jersey Retrons coding for hybrid DNA/RNA molecules
US5436141A (en) 1989-02-24 1995-07-25 University Of Medicine And Dentistry Of New Jersey Method for synthesizing stable single-stranded CDNA in eukaryotes by means of a bacterial retron and products
DK0387775T3 (en) 1989-03-16 1996-11-04 Boehringer Ingelheim Int Genetic unit for inhibiting the function of RNA
US5034323A (en) 1989-03-30 1991-07-23 Dna Plant Technology Corporation Genetic engineering of novel plant phenotypes
US5231020A (en) 1989-03-30 1993-07-27 Dna Plant Technology Corporation Genetic engineering of novel plant phenotypes
AU5630590A (en) 1989-04-05 1990-11-16 United States of America, as represented by the Secretary, U.S. Department of Commerce, The A clone of double-stranded rna virus applied to antibody production, study of retrovirus-like frameshifting and production of proteins in yeast
WO1990012094A1 (en) 1989-04-05 1990-10-18 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce A clone of double-stranded rna virus and applications thereof
EP0473576A4 (en) 1989-05-19 1993-03-10 Hem Research, Inc. Short therapeutic dsrna of defined structure
US5122466A (en) 1989-06-13 1992-06-16 North Carolina State University Ballistic transformation of conifers
GB8916213D0 (en) 1989-07-14 1989-08-31 Ici Plc Dna constructs,cells and plants derived therefrom
HU214927B (en) 1989-08-10 1998-07-28 Plant Genetic Systems N.V. Process for producing plants with modified flower
US5747308A (en) 1989-10-25 1998-05-05 Celltech Therapeutics Limited Recombinant DNA method
US5939600A (en) * 1989-11-03 1999-08-17 Goldbach; Robert Willem Nucleic acids encoding tospovirus genome and expression thereof
HUT57265A (en) * 1989-11-03 1991-11-28 Zaadunie Bv Process for producing plants of diminished infection-sensitivity
US5457189A (en) 1989-12-04 1995-10-10 Isis Pharmaceuticals Antisense oligonucleotide inhibition of papillomavirus
US5578718A (en) * 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
US6245560B1 (en) * 1990-01-18 2001-06-12 The United States Of America As Represented By The Department Of Health And Human Services Vector with multiple target response elements affecting gene expression
JP2746480B2 (en) * 1990-01-18 1998-05-06 アメリカ合衆国 Vectors with multiple target response factors affecting gene expression
US5168064A (en) 1990-04-20 1992-12-01 The Regents Of The University Of California Endo-1,4-β-glucanase gene and its use in plants
GB9009307D0 (en) 1990-04-25 1990-06-20 Ici Plc Dna,constructs,cells and plant derived therefrom
AU7906691A (en) * 1990-05-23 1991-12-10 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Adeno-associated virus (aav)-based eucaryotic vectors
US5270163A (en) * 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
AU8059891A (en) 1990-06-13 1992-01-07 Trustees Of Princeton University, The A (lac) repressor-hsv vp16 chimeric transcriptional activator protein system functioning in transfected mammalian cells
WO1992003454A1 (en) 1990-08-14 1992-03-05 Isis Pharmaceuticals, Inc. Inhibition of influenza virus type a, ann arbor strain h2n2 by antisense oligonucleotides
DE4027616A1 (en) * 1990-08-31 1992-03-05 Boehringer Mannheim Gmbh METHOD FOR DETERMINING POLYMERASE ACTIVITY
MX9100993A (en) 1990-09-10 1992-05-04 Us Agriculture RECOMBINANT SEQUENCE OF DNA ISOLATED AND ENZYME 1-AMINO CYCLOPROPAN-1-CARBOXYL SYNTHESE, RECOMBINANT
US5306862A (en) * 1990-10-12 1994-04-26 Amoco Corporation Method and composition for increasing sterol accumulation in higher plants
SE467358B (en) 1990-12-21 1992-07-06 Amylogene Hb GENETIC CHANGE OF POTATISE BEFORE EDUCATION OF AMYLOPECT TYPE STARCH
SE467160B (en) 1990-12-21 1992-06-01 Amylogene Hb GENETIC MODIFICATION OF POTATOIS BEFORE EDUCATION OF AMYLOST TYPE
JPH06505161A (en) 1991-01-25 1994-06-16 ユナイテッド・ステイツ・バイオケミカル・コーポレイション Regulation of nucleic acid translation
GB9105383D0 (en) 1991-03-14 1991-05-01 Immunology Ltd An immunotherapeutic for cervical cancer
GB9106713D0 (en) 1991-03-28 1991-05-15 Ici Plc Dna,dna constructs,cells and plants derived therefrom
US6005167A (en) 1991-04-16 1999-12-21 Mogen International N.V. Male-sterile plants, method for obtaining male-sterile plants and recombinant DNA for use therein
US5683985A (en) 1991-04-18 1997-11-04 The Salk Institute For Biological Studies Oligonucleotide decoys and methods relating thereto
FR2675803B1 (en) 1991-04-25 1996-09-06 Genset Sa CLOSED, ANTISENSE AND SENSE OLIGONUCLEOTIDES AND THEIR APPLICATIONS.
GB9109063D0 (en) 1991-04-26 1991-06-12 Ici Plc Modification of lignin synthesis in plants
WO1992021757A1 (en) 1991-05-30 1992-12-10 Plant Genetic Systems, N.V. Nematode-responsive plant promoters
CA2112373C (en) 1991-07-11 2010-04-20 Timothy A. Holton Genetic sequences encoding flavonoid pathway enzymes and uses therefor
US6010908A (en) 1992-08-21 2000-01-04 The Regents Of The University Of California Gene therapy by small fragment homologous replacement
US5714323A (en) 1991-08-30 1998-02-03 The University Of Medecine And Dentistry Of New Jersey Over expression of single-stranded molecules
CA2073630C (en) 1991-08-30 2007-12-11 Atsushi Ohshima Method for synthesizing single-stranded stem-loop dnas, the products and uses therefor
AU2928492A (en) 1991-11-20 1993-06-15 Mogen International N.V. A method for obtaining plants with reduced susceptibility to plant-parasitic nematodes
FR2685346B1 (en) * 1991-12-18 1994-02-11 Cis Bio International PROCESS FOR THE PREPARATION OF DOUBLE-STRANDED RNA, AND ITS APPLICATIONS.
ES2166361T3 (en) 1992-02-19 2002-04-16 State Of Oregon Acting By & Th PRODUCTION OF VIRUS RESISTANT PLANTS THROUGH THE INTRODUCTION OF INTRADUCIBLE VIRAL RNA OF POSITIVE SENSE.
DE4208107A1 (en) 1992-03-13 1993-09-16 Bayer Ag PSEUDORABIES VIRUS (PRV) POLYNUCLEOTIDES AND THEIR USE IN THE MANUFACTURE OF VIRUS RESISTANT EUKARYOTIC CELLS
US5593874A (en) * 1992-03-19 1997-01-14 Monsanto Company Enhanced expression in plants
US5496698A (en) * 1992-08-26 1996-03-05 Ribozyme Pharmaceuticals, Inc. Method of isolating ribozyme targets
GB9210273D0 (en) 1992-05-13 1992-07-01 Ici Plc Dna
US5693535A (en) 1992-05-14 1997-12-02 Ribozyme Pharmaceuticals, Inc. HIV targeted ribozymes
US6451603B1 (en) 1992-06-29 2002-09-17 Gene Shears Pty. Limited Ribozyme nucleic acids and methods of use thereof for controlling viral pathogens
ATE171210T1 (en) 1992-07-02 1998-10-15 Hybridon Inc SELF-STABILIZED OLIGONUCLEOTIDES AS THERAPEUTICS
JPH07503376A (en) 1992-07-30 1995-04-13 ケイヘーネ・エヌ・ベー DNA constructs and cells and plants derived therefrom
CA2106260A1 (en) * 1992-09-17 1994-03-18 Robert M. Kotin Human adeno-associated virus integration site dna and uses thereof
WO1994007367A1 (en) 1992-09-29 1994-04-14 Apollon, Inc. Anti-viral oligomers that bind polypurine tracts of single-stranded rna or rna-dna hybrids
AU674029B2 (en) 1992-10-15 1996-12-05 Syngenta Mogen Bv Genetic moderation or restoration of plant phenotypes
US6872872B1 (en) 1992-11-17 2005-03-29 E. I. Du Pont De Nemours And Company Genes for microsomal delta-12 fatty acid desaturases and related enzymes from plants
US6372965B1 (en) 1992-11-17 2002-04-16 E.I. Du Pont De Nemours And Company Genes for microsomal delta-12 fatty acid desaturases and hydroxylases from plants
ZA939767B (en) 1993-01-21 1994-09-14 Univ North Carolina State Nematode-resistant transgenic plants
CA2150133A1 (en) 1993-02-05 1994-08-18 Vincent Jean-Marie Armel Arondel Altered linolenic and linoleic acid content in plants
US6069298A (en) 1993-02-05 2000-05-30 Regents Of The University Of Minnesota Methods and an acetyl CoA carboxylase gene for conferring herbicide tolerance and an alteration in oil content of plants
EP0620281A3 (en) * 1993-03-31 1995-05-03 Mitsubishi Corp Oilseed crops producing valuable seeds having altered amino acid composition and fatty acid composition.
WO1994029465A1 (en) 1993-06-08 1994-12-22 Nunhems Zaden B.V. Process for generating male sterile plants
KR960704034A (en) 1993-07-19 1996-08-31 다니엘 엘. 캐시앙 Enhancement of Oligonucleotide Inhibition of Protein Production, Cell Proliferation, and / or Multiplication of Infectious Disease Pathogens
US5739309A (en) 1993-07-19 1998-04-14 Gen-Probe Incorporated Enhancement of oligonucleotide inhibition of protein production, cell proliferation and / or multiplication of infectious disease pathogens
US5808036A (en) 1993-09-01 1998-09-15 Research Corporation Technologies Inc. Stem-loop oligonucleotides containing parallel and antiparallel binding domains
US5514546A (en) 1993-09-01 1996-05-07 Research Corporation Technologies, Inc. Stem-loop oligonucleotides containing parallel and antiparallel binding domains
ATE400651T1 (en) 1993-09-10 2008-07-15 Univ Columbia USE OF GREEN FLUORESCENT PROTEIN
GB9318927D0 (en) 1993-09-13 1993-10-27 Zeneca Ltd Regulation of senescence
CA2172153C (en) 1993-09-20 2010-03-09 John C. Reed Regulation of bcl-2 gene expression
US5858981A (en) 1993-09-30 1999-01-12 University Of Pennsylvania Method of inhibiting phagocytosis
GB9320548D0 (en) 1993-10-06 1993-11-24 Sandoz Ltd Improvements in or relating to organic compounds
US5624803A (en) 1993-10-14 1997-04-29 The Regents Of The University Of California In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom
US5801154A (en) * 1993-10-18 1998-09-01 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of multidrug resistance-associated protein
US5578716A (en) 1993-12-01 1996-11-26 Mcgill University DNA methyltransferase antisense oligonucleotides
US5908779A (en) 1993-12-01 1999-06-01 University Of Connecticut Targeted RNA degradation using nuclear antisense RNA
FR2714383B1 (en) 1993-12-29 1996-02-09 Centre Nat Rech Scient Control of gene expression.
JP3691849B2 (en) 1994-01-05 2005-09-07 ジーン・シアーズ・ピーティーワイ・リミテッド Ribozymes targeting retroviral packaging sequences, expression constructs thereof, and recombinant retroviruses containing such constructs
US5849991A (en) * 1994-01-27 1998-12-15 Bresatch Limited Mice homozygous for an inactivated α 1,3-galactosyl transferase gene
AU706417B2 (en) 1994-02-23 1998-06-17 Ribozyme Pharmaceuticals, Inc. Method and reagent for inhibiting the expression of disease related genes
US5686649A (en) 1994-03-22 1997-11-11 The Rockefeller University Suppression of plant gene expression using processing-defective RNA constructs
US5631148A (en) 1994-04-22 1997-05-20 Chiron Corporation Ribozymes with product ejection by strand displacement
US6054299A (en) * 1994-04-29 2000-04-25 Conrad; Charles A. Stem-loop cloning vector and method
US6146886A (en) 1994-08-19 2000-11-14 Ribozyme Pharmaceuticals, Inc. RNA polymerase III-based expression of therapeutic RNAs
EP0772678A4 (en) 1994-09-12 1999-10-20 Hope City Modulation of drug radiation resistant genes
US5576716A (en) * 1994-12-07 1996-11-19 Sadler; Kermit M. Owner oriented system for locating lost or stolen property
AU5741396A (en) 1995-05-11 1996-11-29 Hybridon, Inc. Pyrimidine targeting hairpin triplex-forming oligonucleotide s
US5691140A (en) 1995-05-18 1997-11-25 New England Biolabs, Inc. Bidirectional in vitro transcription vectors utilizing a single RNA polymerase for both directions
US5693773A (en) 1995-06-07 1997-12-02 Hybridon Incorporated Triplex-forming antisense oligonucleotides having abasic linkers targeting nucleic acids comprising mixed sequences of purines and pyrimidines
EP0837624A1 (en) 1995-06-30 1998-04-29 DNA Plant Technology Corporation Delayed ripening tomato plants
FR2737501B1 (en) * 1995-07-31 1997-10-24 Transgene Sa NOVEL AUXILIARY VIRUSES FOR THE PREPARATION OF RECOMBINANT VIRAL VECTORS
AU6904596A (en) 1995-09-13 1997-04-01 Chiron Corporation Method and construct for screening for inhibitors of transcriptional activation
EP0851919A1 (en) 1995-09-20 1998-07-08 University of Massachusetts Worcester Antisense oligonucleotide chemotherapy for benign hyperplasia or cancer of the prostate
PL187026B1 (en) 1995-10-06 2004-04-30 Plant Genetic Systems Nv Seeds dropping
JPH09110894A (en) 1995-10-17 1997-04-28 Soyaku Gijutsu Kenkyusho:Kk Hybric dna/rna oligonucleotide and antiviral agent
EP0771878A1 (en) 1995-10-31 1997-05-07 Plant Genetic Systems N.V. Plants with reduced glucosinolate content
US5773692A (en) 1995-12-12 1998-06-30 Her Majesty The Queen In Right Of Canada, As Represented By Agriculture And Agri-Food Canada Anti-sense RNA for CAB transcript to reduce chlorophyll content in plants
CA2190304A1 (en) 1995-12-15 1997-06-16 Elazar Rabbani Property effecting and/or property exhibiting compositions for therapeutic and diagnostic uses
IT1283876B1 (en) 1996-01-12 1998-05-07 Univ Roma CHIMERIC RIBOZYME-SNRNA MOLECULES WITH CATALYTIC ACTIVITY FOR NUCLEAR-LOCATED RNA
EP0880598A4 (en) * 1996-01-23 2005-02-23 Affymetrix Inc Nucleic acid analysis techniques
US5891855A (en) * 1996-02-12 1999-04-06 The Scripps Research Institute Inhibitors of leaderless protein export
JP3847366B2 (en) 1996-02-22 2006-11-22 アンジェスMg株式会社 Stationary mitotic cell growth agent using antisense oligonucleotide
US5989864A (en) * 1996-10-29 1999-11-23 Smithkline Beecham Corporation DNA encoding spo-rel polypeptides
WO1997034003A1 (en) 1996-03-13 1997-09-18 National Research Council Of Canada Process of raising squalene levels in plants and dna sequences used therefor
DK0888452T3 (en) * 1996-03-15 2004-06-14 Munin Corp Human CYR61, an extracellular matrix signaling molecule
US6022863A (en) 1996-05-21 2000-02-08 Yale University Regulation of gene expression
TR199802421T2 (en) 1996-05-24 1999-02-22 Biogen , Inc. Substances that regulate tissue regeneration.
US5994526A (en) 1996-06-21 1999-11-30 Plant Genetic Systems Gene expression in plants
CA2255057A1 (en) 1996-06-21 1997-12-31 Plant Genetic Systems, N.V. Gene expression in plants
US5952546A (en) * 1996-06-27 1999-09-14 Dna Plant Technology Corporation Delayed ripening tomato plants with T-DNA bearing a truncated ACC2 synthase gene
US5850026A (en) 1996-07-03 1998-12-15 Cargill, Incorporated Canola oil having increased oleic acid and decreased linolenic acid content
JP2001503735A (en) 1996-07-03 2001-03-21 ユニバーシティ オブ ピッツバーグ Emulsion formulation for hydrophilic active reagent
IL128124A0 (en) * 1996-08-02 1999-11-30 Genesense Technologies Inc Antitumor antisense sequences directed against r1 and r2 components of ribonucleotide reductase
DE19631919C2 (en) 1996-08-07 1998-07-16 Deutsches Krebsforsch Anti-sense RNA with secondary structure
US5747338A (en) 1996-08-15 1998-05-05 Chiron Corporation Method and construct for screening for inhibitors of transcriptional activation
US6225290B1 (en) 1996-09-19 2001-05-01 The Regents Of The University Of California Systemic gene therapy by intestinal cell transformation
US5814500A (en) 1996-10-31 1998-09-29 The Johns Hopkins University School Of Medicine Delivery construct for antisense nucleic acids and methods of use
GB9703146D0 (en) 1997-02-14 1997-04-02 Innes John Centre Innov Ltd Methods and means for gene silencing in transgenic plants
JP2001514491A (en) 1997-02-21 2001-09-11 ダニスコ エイ/エス Antisense intron inhibition of starch branching enzyme expression
CA2254785A1 (en) * 1997-03-10 1998-09-17 Japan Tobacco Inc. Antisense base sequences
GB9706381D0 (en) 1997-03-27 1997-05-14 Cambridge Advanced Tech Improvements relating to the specificity of gene expression
US7589253B2 (en) 1997-04-15 2009-09-15 Commonwealth Scientific And Industrial Research Organisation Fatty acid epoxygenase genes from plants and uses therefor in modifying fatty acid metabolism
US5942395A (en) 1997-05-09 1999-08-24 Universite De Montreal Hybrid ribozymes and methods of use
CA2300938A1 (en) * 1997-08-20 1999-02-25 Somagenics, Inc. Antisense and antigene therapeutics with improved binding properties and methods for their use
GB9720148D0 (en) 1997-09-22 1997-11-26 Innes John Centre Innov Ltd Gene silencing materials and methods
EP1032692A1 (en) 1997-11-18 2000-09-06 Pioneer Hi-Bred International, Inc. Targeted manipulation of herbicide-resistance genes in plants
DE19754622A1 (en) 1997-12-09 1999-06-10 Antje Von Dr Schaewen Plant GntI sequences and the use thereof for obtaining plants with reduced or missing N-acetylglucosaminyltransferase I (GnTI) activity
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
AUPP249298A0 (en) 1998-03-20 1998-04-23 Ag-Gene Australia Limited Synthetic genes and genetic constructs comprising same I
WO1999049029A1 (en) 1998-03-20 1999-09-30 Benitec Australia Ltd Control of gene expression
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
EP1068311B2 (en) 1998-04-08 2020-12-09 Commonwealth Scientific and Industrial Research Organisation Methods and means for obtaining modified phenotypes
US20040214330A1 (en) 1999-04-07 2004-10-28 Waterhouse Peter Michael Methods and means for obtaining modified phenotypes
EP0959133A1 (en) 1998-05-22 1999-11-24 Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) A process for inhibiting expression of genes
AR020078A1 (en) 1998-05-26 2002-04-10 Syngenta Participations Ag METHOD FOR CHANGING THE EXPRESSION OF AN OBJECTIVE GENE IN A PLANT CELL
GB9827152D0 (en) 1998-07-03 1999-02-03 Devgen Nv Characterisation of gene function using double stranded rna inhibition
US6040908A (en) * 1998-07-28 2000-03-21 Litton Systems, Inc. Method for stress tuning fiber optic sensor coils
CA2361201A1 (en) 1999-01-28 2000-08-03 Medical College Of Georgia Research Institute, Inc. Composition and method for in vivo and in vitro attenuation of gene expression using double stranded rna
DE19956568A1 (en) 1999-01-30 2000-08-17 Roland Kreutzer Method and medicament for inhibiting the expression of a given gene
ES2316363T3 (en) 1999-04-20 2009-04-16 Bayer Bioscience N.V. METHODS FOR ADMINISTRATION OF RNA INHIBITOR TO THE PLANTS AND APPLICATIONS OF THE SAME.
WO2000063364A2 (en) 1999-04-21 2000-10-26 American Home Products Corporation Methods and compositions for inhibiting the function of polynucleotide sequences
US20040138168A1 (en) 1999-04-21 2004-07-15 Wyeth Methods and compositions for inhibiting the function of polynucleotide sequences
JP2003501102A (en) 1999-06-14 2003-01-14 エクセリクシス,インク Animal models and methods for the analysis of lipid metabolism and the screening of pharmaceuticals and insecticides that regulate lipid metabolism
IL147026A0 (en) 1999-07-09 2002-08-14 American Home Prod Method and compositions for preventing the formation of aberrant rna during transcripting of a plasmid sequence
US6423885B1 (en) 1999-08-13 2002-07-23 Commonwealth Scientific And Industrial Research Organization (Csiro) Methods for obtaining modified phenotypes in plant cells
AU7487300A (en) 1999-09-16 2001-04-17 Genoptera, Llc Lethal insectidice targets
AU1086501A (en) 1999-10-15 2001-04-30 Carnegie Institution Of Washington Rna interference pathway genes as tools for targeted genetic interference
US6291504B1 (en) 1999-10-20 2001-09-18 Dupont Pharmaceuticals Company Acylsemicarbazides and their uses
GB9925459D0 (en) 1999-10-27 1999-12-29 Plant Bioscience Ltd Gene silencing
GB9927444D0 (en) 1999-11-19 2000-01-19 Cancer Res Campaign Tech Inhibiting gene expression
AU2047001A (en) 1999-11-24 2001-06-04 Dna Plant Technology Corporation Methods of inhibiting plant parasitic nematodes and insect pests by expression of nematode and insect specific double-stranded rna in plants
WO2001038359A2 (en) 1999-11-29 2001-05-31 Genoptera, Llc Drosophila nicotinic acetylcholine receptor
GB9930691D0 (en) 1999-12-24 2000-02-16 Devgen Nv Improvements relating to double-stranded RNA inhibition
CN1426466A (en) 2000-03-17 2003-06-25 贝尼泰克澳大利亚有限公司 Genetic silencing
AU2001249622B2 (en) 2000-03-30 2007-06-07 Massachusetts Institute Of Technology RNA sequence-specific mediators of RNA interference
GB2362383B (en) 2000-05-19 2003-12-31 Devgen Nv Gene expression system
US6995258B1 (en) * 2000-05-25 2006-02-07 City Of Hope Nucleolar targeting of therapeutics against HIV
HUP0302320A3 (en) 2000-06-23 2005-11-28 Pioneer Hi Bred Int Recombinant constructs and their use in reducing gene expression
US7109393B2 (en) 2000-08-15 2006-09-19 Mendel Biotechnology, Inc. Methods of gene silencing using inverted repeat sequences
ES2728168T3 (en) 2000-12-01 2019-10-22 Max Planck Gesellschaft Small RNA molecules that mediate RNA interference
US20020150968A1 (en) * 2001-01-10 2002-10-17 Wang Peng G. Glycoconjugate and sugar nucleotide synthesis using solid supports
CA2369944A1 (en) 2001-01-31 2002-07-31 Nucleonics Inc. Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell
AUPR621501A0 (en) 2001-07-06 2001-08-02 Commonwealth Scientific And Industrial Research Organisation Delivery of ds rna
US7691995B2 (en) 2001-07-12 2010-04-06 University Of Massachusetts In vivo production of small interfering RNAS that mediate gene silencing
WO2003023015A2 (en) 2001-09-13 2003-03-20 California Institute Of Technology Method for expression of small antiviral rna molecules within a cell
JP2005502383A (en) 2001-09-27 2005-01-27 ティモシー アルバート ホルトン Method for preparing a nucleic acid library
US20030148519A1 (en) * 2001-11-14 2003-08-07 Engelke David R. Intracellular expression and delivery of siRNAs in mammalian cells
GB0130955D0 (en) 2001-12-24 2002-02-13 Cancer Res Ventures Expression system
US20040022748A1 (en) 2002-03-12 2004-02-05 Unilever Home & Personal Care Usa, Division Of Conopco, Inc. Method of enhancing skin lightening
BR0308424A (en) 2002-03-14 2005-02-22 Commw Scient Ind Res Org Methods and means for efficiently downregulating the expression of any gene of interest in eukaryotic cells and organisms
ITRM20020253A1 (en) 2002-05-08 2003-11-10 Univ Roma SNRNA CHEMICAL MOLECULES WITH ANTISENSE SEQUENCES FOR SPLICING JUNCTIONS OF THE DYSTROPHINE GENE AND THERAPEUTIC APPLICATIONS.
US20040106566A1 (en) * 2002-05-17 2004-06-03 Shi-Lung Lin RNA-splicing and processing-directed gene silencing and the relative applications thereof
US20040234504A1 (en) * 2002-12-18 2004-11-25 Verma Inder M. Methods of inhibiting gene expression by RNA interference
ATE528402T1 (en) 2003-02-19 2011-10-15 Commw Scient Ind Res Org EFFICIENT GENE silencing in plants using short DSRNA sequences
US20080044906A1 (en) 2003-09-12 2008-02-21 Peter Michael Waterhouse Modified Gene-Silencing Nucleic Acid Molecules and Uses Thereof
CA2650861A1 (en) 2006-05-03 2007-11-15 Commonwealth Scientific And Industrial Research Organisation Improved gene silencing methods
US9642116B2 (en) * 2008-03-05 2017-05-02 Nxp B.V. Dynamic MBSFN area configuration method in consideration of radio resource efficiency and system thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998053083A1 (en) * 1997-05-21 1998-11-26 Zeneca Limited Gene silencing

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ASSAAD F F, TUCKER K L, SIGNER E R: "EPIGENETIC REPEAT-INDUCED GENE SILENCING (RIGS) IN ARABIDOPSIS", PLANT MOLECULAR BIOLOGY, SPRINGER, DORDRECHT., NL, vol. 22, no. 06, 1 January 1993 (1993-01-01), NL, pages 1067 - 1085, XP000982135, ISSN: 0167-4412, DOI: 10.1007/BF00028978 *
DORER D R, HENIKHOFF S: "TRANSGENE REPEAT ARRAYS INTERACT WITH DISTANT HETEROCHROMATIN AND CAUSE SILENCING IN CIS AND TRANS", GENETICS, GENETICS SOCIETY OF AMERICA, AUSTIN, TX, US, vol. 147, no. 03, 1 November 1997 (1997-11-01), US, pages 1181 - 1190, XP000987071, ISSN: 0016-6731 *
DORER D R, HENIKOFF S: "EXPANSIONS OF TRANSGENE REPEATS CAUSE HETEROCHROMATIN FORMATION ANDGENE SILENCING IN DROSOPHILA", CELL, CELL PRESS, US, vol. 77, no. 07, 1 July 1994 (1994-07-01), US, pages 993 - 1002, XP000982138, ISSN: 0092-8674, DOI: 10.1016/0092-8674(94)90439-1 *
GRANT S R: "DISSECTING THE MECHANISMS OF POSTTRANSCRIPTIONAL GENE SILENCING: DIVIDE AND CONQUER", CELL, CELL PRESS, US, vol. 96, no. 03, 5 February 1999 (1999-02-05), US, pages 303 - 306, XP000982139, ISSN: 0092-8674, DOI: 10.1016/S0092-8674(00)80541-3 *
HAMILTON ANDREW J ET AL: "A transgene with repeated DNA causes high frequency, post-transcriptional suppression of ACC-oxidase gene expression in tomato.", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD., GB, vol. 15, no. 6, 1 January 1998 (1998-01-01), GB, pages 737 - 746, XP002146504, ISSN: 0960-7412, DOI: 10.1046/j.1365-313X.1998.00251.x *
QUE Q, JORGENSEN R A: "HOMOLOGY-BASED CONTROL OF GENE EXPRESSION PATTERNS IN TRANSGENIC PETUNIA FLOWERS", DEVELOPMENTAL GENETICS, NEW-YORK, NY., US, vol. 22, no. 01, 1 January 1998 (1998-01-01), US, pages 100 - 109, XP000987329, ISSN: 0192-253X, DOI: 10.1002/(SICI)1520-6408(1998)22:1<100::AID-DVG10>3.0.CO;2-D *
SIJEN T, ET AL.: "RNA-MEDIATED VIRUS RESISTANCE: ROLE OF REPEATED TRANSGENES AND DELINEATION OF TARGETED REGIONS", THE PLANT CELL, AMERICAN SOCIETY OF PLANT BIOLOGISTS, US, vol. 08, 1 December 1996 (1996-12-01), US, pages 2277 - 2294, XP000982134, ISSN: 1040-4651, DOI: 10.1105/tpc.8.12.2277 *
STAM M, MOL J N M, KOOTER J M: "THE SILENCE OF GENES IN TRANSGENIC PLANTS", ANNALS OF BOTANY., ACADEMIC PRESS, LONDON., GB, vol. 79, no. 01, 1 January 1997 (1997-01-01), GB, pages 03 - 12, XP000987302, ISSN: 0305-7364, DOI: 10.1006/anbo.1996.0295 *

Cited By (442)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9102939B2 (en) 1997-12-23 2015-08-11 The Carnegie Institution Of Washington Genetic inhibition by double-stranded RNA
US9029527B2 (en) 1998-03-20 2015-05-12 Commonwealth Scientific And Industrial Research Organisation Synthetic genes and genetic constructs
US9963698B2 (en) 1998-03-20 2018-05-08 Commonwealth Scientific And Industrial Research Organisation Control of gene expression
EP2267139A3 (en) * 1998-04-08 2011-05-25 Commonwealth Scientific and Industrial Research Organization Methods and means for obtaining modified phenotypes
EP1068311B2 (en) 1998-04-08 2020-12-09 Commonwealth Scientific and Industrial Research Organisation Methods and means for obtaining modified phenotypes
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
EP3214177A3 (en) * 1998-04-08 2017-11-22 Commonwealth Scientific and Industrial Research Organisation Methods and means for obtaining modified phenotypes
US9441239B2 (en) 1998-04-08 2016-09-13 Commonwealth Scientific & Industrial Research Organisation Methods and means for obtaining modified phenotypes
EP1516931A2 (en) 1998-05-26 2005-03-23 Syngenta Participations AG DSRNA-mediated regulation of gene expression in plants
JP2009261403A (en) * 1998-05-26 2009-11-12 Syngenta Participations Ag Dsrna-mediated regulation of gene expression in plant
EP1516931A3 (en) * 1998-05-26 2007-10-31 Syngenta Participations AG DSRNA-mediated regulation of gene expression in plants
US9902955B2 (en) 1999-01-30 2018-02-27 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
EP1798285A1 (en) * 1999-01-30 2007-06-20 Alnylam Europe AG Methods and medicament for inhibition the expression of a defined gene
US9133454B2 (en) 1999-01-30 2015-09-15 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
EP3018207A1 (en) * 1999-01-30 2016-05-11 Alnylam Europe AG Oligoribonucucleotide for inhibiting the expression of a predefined gene
AU2005201044B2 (en) * 1999-01-30 2008-05-29 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a defined gene
DE10066235C5 (en) 1999-01-30 2014-08-21 Alnylam Europe Ag Method and medicament for inhibiting the expression of a given gene
EP1550719A1 (en) * 1999-01-30 2005-07-06 Alnylam Europe AG Methods and medicament for inhibition the expression of a defined gene
AU781598B2 (en) * 1999-04-21 2005-06-02 Alnylam Pharmaceuticals, Inc. Methods and compositions for inhibiting the function of polynucleotide sequences
EP1801215A3 (en) * 1999-05-10 2008-12-17 Syngeta Participations AG Regulation of viral gene expression
EP1801215A2 (en) * 1999-05-10 2007-06-27 Syngeta Participations AG Regulation of viral gene expression
CN100420748C (en) * 1999-05-10 2008-09-24 辛根塔参与股份公司 Regulation of viral gene expression
WO2000068374A1 (en) * 1999-05-10 2000-11-16 Syngenta Participations Ag Regulation of viral gene expression
JP2002543783A (en) * 1999-05-10 2002-12-24 シンジェンタ・パティシペーションズ・アクチェンゲゼルシャフト Regulation of viral gene expression
US9708621B2 (en) 1999-08-13 2017-07-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US10190127B2 (en) 1999-08-13 2019-01-29 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US7148336B2 (en) 1999-08-26 2006-12-12 Calgene Llc Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acid levels
US7531718B2 (en) 1999-08-26 2009-05-12 Monsanto Technology, L.L.C. Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
US7256329B2 (en) 1999-08-26 2007-08-14 Calgene Llc Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
US8097778B2 (en) 1999-08-26 2012-01-17 Monsanto Company Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
US7563949B2 (en) 1999-08-26 2009-07-21 Monsanto Technology Llc Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
US7067722B2 (en) 1999-08-26 2006-06-27 Monsanto Technology Llc Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acids
JP2012085641A (en) * 1999-11-19 2012-05-10 Cancer Research Technology Ltd Inhibition of gene expression with dsrna
JP2015109847A (en) * 1999-11-19 2015-06-18 キャンサー・リサーチ・テクノロジー・リミテッドCan Inhibiting gene expression with dsrna
US7829693B2 (en) 1999-11-24 2010-11-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
US7732417B2 (en) 2000-03-16 2010-06-08 Cold Spring Harbor Laboratory Methods and compositions for RNA interference using recombinant Dicer and Argonaut
US8202846B2 (en) 2000-03-16 2012-06-19 Cold Spring Harbor Laboratory Methods and compositions for RNA interference
US8383599B2 (en) 2000-03-16 2013-02-26 Cold Spring Harbor Laboratory Methods and compositions for RNA interference
US8153776B2 (en) 2000-03-16 2012-04-10 Cold Spring Harbor Laboratory Methods and compositions for RNA interference
US8552171B2 (en) 2000-03-30 2013-10-08 University Of Massachusetts RNA sequence-specific mediators of RNA interference
US8790922B2 (en) 2000-03-30 2014-07-29 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA sequence-specific mediators of RNA interference
US8394628B2 (en) 2000-03-30 2013-03-12 University Of Massachusetts RNA sequence-specific mediators of RNA interference
US8420391B2 (en) 2000-03-30 2013-04-16 University Of Massachusetts RNA sequence-specific mediators of RNA interference
US8742092B2 (en) 2000-03-30 2014-06-03 University Of Massachusetts RNA sequence-specific mediators of RNA interference
US10472625B2 (en) 2000-03-30 2019-11-12 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA sequence-specific mediators of RNA interference
US9012621B2 (en) 2000-03-30 2015-04-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA sequence-specific mediators of RNA interference
US9012138B2 (en) 2000-03-30 2015-04-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA sequence-specific mediators of RNA interference
US9193753B2 (en) 2000-03-30 2015-11-24 University Of Massachusetts RNA sequence-specific mediators of RNA interference
US8632997B2 (en) 2000-03-30 2014-01-21 University Of Massachusetts RNA sequence-specific mediators of RNA interference
US9309520B2 (en) 2000-08-21 2016-04-12 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
US7662951B2 (en) 2000-08-30 2010-02-16 Sirna Therapeutics, Inc. RNA interference mediated treatment of Alzheimer's disease using short interfering nucleic acid (siNA)
US8017765B2 (en) 2000-08-30 2011-09-13 Merck Sharp & Dohme Corp. RNA interference mediated treatment of alzheimer's disease using short interfering nucleic acid (siNA)
US6777588B2 (en) 2000-10-31 2004-08-17 Peter Waterhouse Methods and means for producing barley yellow dwarf virus resistant cereal plants
US7078196B2 (en) 2000-12-01 2006-07-18 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften, E.V. RNA interference mediating small RNA molecules
US8445237B2 (en) 2000-12-01 2013-05-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8895721B2 (en) 2000-12-01 2014-11-25 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8362231B2 (en) 2000-12-01 2013-01-29 Max-Planck-Gesellschaft zur Föderung der Wissenschaften E.V. RNA interference mediating small RNA molecules
US8778902B2 (en) 2000-12-01 2014-07-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8796016B2 (en) 2000-12-01 2014-08-05 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8895718B2 (en) 2000-12-01 2014-11-25 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8372968B2 (en) 2000-12-01 2013-02-12 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8993745B2 (en) 2000-12-01 2015-03-31 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8933044B2 (en) 2000-12-01 2015-01-13 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US7056704B2 (en) 2000-12-01 2006-06-06 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. RNA interference mediating small RNA molecules
US8329463B2 (en) 2000-12-01 2012-12-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8853384B2 (en) 2000-12-01 2014-10-07 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US8765930B2 (en) 2000-12-01 2014-07-01 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RNA interference mediating small RNA molecules
US10633656B2 (en) 2000-12-01 2020-04-28 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. RNA interference mediating small RNA molecules
US8945884B2 (en) 2000-12-11 2015-02-03 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites
US9074213B2 (en) 2001-01-09 2015-07-07 Alnylam Pharmacuticals, Inc. Compositions and methods for inhibiting expression of a target gene
US7767802B2 (en) 2001-01-09 2010-08-03 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
US7473525B2 (en) 2001-01-09 2009-01-06 Alnylam Europe Ag Compositions and methods for inhibiting expression of anti-apoptotic genes
US9587240B2 (en) 2001-01-09 2017-03-07 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
US7868160B2 (en) 2001-01-09 2011-01-11 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
US7846718B2 (en) 2001-01-26 2010-12-07 Commonwealth Scientific And Industrial Research Organisation Methods and means for producing efficient silencing construct using recombinational cloning
US6933146B2 (en) 2001-01-26 2005-08-23 Commonwealth Scientific And Industrial Research Corporation Methods and means for producing efficient silencing construct using recombinational cloning
US8877435B2 (en) 2001-01-26 2014-11-04 Commonwealth Scientific And Industrial Research Organisation Methods and means for producing efficient silencing construct using recombinational cloning
US7732660B2 (en) 2001-01-26 2010-06-08 Commonwealth Scientific And Industrial Research Corporation Methods and means for producing efficient silencing construct using recombinational cloning
US9051566B2 (en) 2001-01-31 2015-06-09 Alnylam Pharmaceuticals, Inc. Post-transcriptional gene silencing using expressed double stranded RNA
US7022828B2 (en) 2001-04-05 2006-04-04 Sirna Theraputics, Inc. siRNA treatment of diseases or conditions related to levels of IKK-gamma
US7858625B2 (en) 2001-05-18 2010-12-28 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US7491805B2 (en) 2001-05-18 2009-02-17 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US7964578B2 (en) 2001-05-18 2011-06-21 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US8017761B2 (en) 2001-05-18 2011-09-13 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of Stearoyl-CoA desaturase (SCD) gene expression using short interfering nucelic acid (siNA)
US7659389B2 (en) 2001-05-18 2010-02-09 Sirna Therapeutics, Inc. RNA interference mediated inhibition of MYC and/or MYB gene expression using short interfering nucleic acid (siNA)
US7833992B2 (en) 2001-05-18 2010-11-16 Merck Sharpe & Dohme Conjugates and compositions for cellular delivery
US9994853B2 (en) 2001-05-18 2018-06-12 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
US8008472B2 (en) 2001-05-29 2011-08-30 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of human immunodeficiency virus (HIV) gene expression using short interfering nucleic acid (siNA)
US9663786B2 (en) 2001-07-06 2017-05-30 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
EP2333061A1 (en) 2001-07-06 2011-06-15 Commonwealth Scientific and Industrial Research Organization Delivery of dsRNA to arthropods
US8263573B2 (en) 2001-07-06 2012-09-11 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US8101343B2 (en) 2001-07-06 2012-01-24 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US8877727B2 (en) 2001-07-06 2014-11-04 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US8415320B2 (en) 2001-07-06 2013-04-09 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US10323245B2 (en) 2001-07-06 2019-06-18 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US9085770B2 (en) 2001-07-06 2015-07-21 Commonwealth Scientific And Industrial Research Organisation Delivery of dsRNA to arthropods
US8232260B2 (en) 2001-07-12 2012-07-31 University Of Massachusetts In vivo production of small interfering RNAs that mediate gene silencing
US7691995B2 (en) 2001-07-12 2010-04-06 University Of Massachusetts In vivo production of small interfering RNAS that mediate gene silencing
US10731155B2 (en) 2001-07-12 2020-08-04 University Of Massachusetts In vivo production of small interfering RNAs that mediate gene silencing
US8557785B2 (en) * 2001-07-12 2013-10-15 University Of Massachusetts In vivo production of small interfering RNAS that mediate gene silencing
US20180163206A1 (en) * 2001-07-12 2018-06-14 University Of Massachusetts In vivo production of small interfering rnas that mediate gene silencing
US9175287B2 (en) 2001-07-12 2015-11-03 University Of Massachusetts In vivo production of small interfering RNAs that mediate gene silencing
US8530438B2 (en) * 2001-07-12 2013-09-10 University Of Massachusetts Vivo production of small interfering RNAs that mediate gene silencing
US7893036B2 (en) 2001-07-12 2011-02-22 University Of Massachusetts In vivo production of small interfering RNAs that mediate gene silencing
US9850487B2 (en) 2001-07-12 2017-12-26 University Of Massachusetts In vivo production of small interfering RNAs that mediate gene silencing
US10590418B2 (en) 2001-07-23 2020-03-17 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals
US9018179B2 (en) 2001-07-23 2015-04-28 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals
US10517887B2 (en) 2001-07-23 2019-12-31 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals
US8614370B2 (en) 2001-07-24 2013-12-24 Monsanto Technology Llc Nucleic acid sequences from Diabrotica virgifera virgifera leconte and uses thereof
US7612194B2 (en) 2001-07-24 2009-11-03 Monsanto Technology Llc Nucleic acid sequences from Diabrotica virgifera virgifera LeConte and uses thereof
US8829264B2 (en) 2002-01-22 2014-09-09 Cold Spring Harbor Laboratory Methods and compositions for RNA interference
US9796978B1 (en) 2002-02-01 2017-10-24 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US10196640B1 (en) 2002-02-01 2019-02-05 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US10036025B2 (en) 2002-02-01 2018-07-31 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US10106793B2 (en) 2002-02-01 2018-10-23 Life Technologies Corporation Double-stranded oligonucleotides
US9777275B2 (en) 2002-02-01 2017-10-03 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US10626398B2 (en) 2002-02-01 2020-04-21 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US9592250B2 (en) 2002-02-01 2017-03-14 Life Technologies Corporation Double-stranded oligonucleotides
US8846894B2 (en) 2002-02-20 2014-09-30 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US8153778B2 (en) 2002-02-20 2012-04-10 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of vascular cell adhesion molecule (VCAM) gene expression using short interfering nucleic acid (siNA)
EP2287306A1 (en) 2002-02-20 2011-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US7897756B2 (en) 2002-02-20 2011-03-01 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering nucleic acid (siNA)
US7897752B2 (en) 2002-02-20 2011-03-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of telomerase gene expression using short interfering nucleic acid (siNA)
US7897753B2 (en) 2002-02-20 2011-03-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of XIAP gene expression using short interfering nucleic acid (siNA)
US7897757B2 (en) 2002-02-20 2011-03-01 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of protein tyrosine phosphatase-1B (PTP-1B) gene expression using short interfering nucleic acid (siNA)
US7897755B2 (en) 2002-02-20 2011-03-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)
US7858771B2 (en) 2002-02-20 2010-12-28 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of muscarinic colinergic receptor gene expression using short interfering nucleic acid (siNA)
WO2003070914A2 (en) * 2002-02-20 2003-08-28 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF FOS GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US8273866B2 (en) 2002-02-20 2012-09-25 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
WO2003074654A2 (en) * 2002-02-20 2003-09-12 Sirna Therapeurics, Inc Rna interference mediated inhibition of gene expression using short interfering nucleic acid (sina)
US7910724B2 (en) 2002-02-20 2011-03-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Fos gene expression using short interfering nucleic acid (siNA)
US7910725B2 (en) 2002-02-20 2011-03-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (siNA)
US7915400B2 (en) 2002-02-20 2011-03-29 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of hepatitis C virus (HCV) gene expression using short interfering nucleic acid (siNA)
US7691999B2 (en) 2002-02-20 2010-04-06 Sirna Therapeutics, Inc. RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering nucleic acid (siNA)
WO2003070914A3 (en) * 2002-02-20 2003-12-24 Sirna Therapeutics Inc RNA INTERFERENCE MEDIATED INHIBITION OF FOS GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7923549B2 (en) 2002-02-20 2011-04-12 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (siNA)
WO2003074654A3 (en) * 2002-02-20 2004-02-05 Sirna Therapeurics Inc Rna interference mediated inhibition of gene expression using short interfering nucleic acid (sina)
US7928219B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of placental growth factor gene expression using short interfering nucleic acid (SINA)
US7928220B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of stromal cell-derived factor-1 (SDF-1) gene expression using short interfering nucleic acid (siNA)
US7928218B2 (en) 2002-02-20 2011-04-19 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of polycomb group protein EZH2 gene expression using short interfering nucleic acid (siNA)
US7935812B2 (en) 2002-02-20 2011-05-03 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of hepatitis C virus (HCV) expression using short interfering nucleic acid (siNA)
EP3459963A1 (en) 2002-02-20 2019-03-27 Sirna Therapeutics, Inc. Rna interference mediated inhibition of gene expression using short interfering nucleic acid (sina)
US7943757B2 (en) 2002-02-20 2011-05-17 Mcswiggen James RNA interference mediated inhibition of intercellular adhesion molecule (ICAM) gene expression using short interfering nucleic acid (siNA)
US7176304B2 (en) 2002-02-20 2007-02-13 Mcswiggen James RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US7855284B2 (en) 2002-02-20 2010-12-21 Sirna Therapeutics, Inc. RNA interference mediated inhibition of checkpoint kinase-1 (CHK-1) gene expression using short interfering nucleic acid (siNA)
WO2003070972A3 (en) * 2002-02-20 2004-06-03 Ribozyme Pharm Inc RNA INTERFERENCE MEDIATED INHIBITION OF CHROMOSOME TRANSLOCATION GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7956178B2 (en) 2002-02-20 2011-06-07 Mcswiggen James RNA interference mediated inhibition of GRB2 associated binding protein (GAB2) gene expression using short interfering nucleic acid (siNA)
GB2396616A (en) * 2002-02-20 2004-06-30 Sirna Therapeutics Inc RNA interference mediated inhibition of gene expression using short interfering nucleic acid (SINA)
US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
WO2005028649A1 (en) 2002-02-20 2005-03-31 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
GB2396616B (en) * 2002-02-20 2005-09-21 Sirna Therapeutics Inc RNA interference mediated inhibition of gene expression using double stranded short interfering nucleic acid (SINA)
US8258288B2 (en) 2002-02-20 2012-09-04 Sirna Therapeutics, Inc. RNA interference mediated inhibition of respiratory syncytial virus (RSV) expression using short interfering nucleic acid (siNA)
EP2902406A1 (en) 2002-02-20 2015-08-05 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
WO2003070193A3 (en) * 2002-02-20 2006-08-24 James Mcswiggen RNA interference mediated inhibition of HIV gene expression using short interfering nucleic acid (siNA)
US7977472B2 (en) 2002-02-20 2011-07-12 Leonid Beigelman RNA interference mediated inhibition of myostatin gene expression using short interfering nucleic acid (siNA)
US8202979B2 (en) 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
US7678897B2 (en) 2002-02-20 2010-03-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)
EP2287305A1 (en) 2002-02-20 2011-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
WO2003070966A3 (en) * 2002-02-20 2004-03-11 Sirna Therapeutics Inc RNA INTERFERENCE MEDIATED TARGET DISCOVERY AND TARGET VALIDATION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7893248B2 (en) 2002-02-20 2011-02-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Myc and/or Myb gene expression using short interfering nucleic acid (siNA)
US9657294B2 (en) 2002-02-20 2017-05-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US8008473B2 (en) 2002-02-20 2011-08-30 Mcswiggen James RNA interference mediated inhibition of TNF and TNF receptor gene expression using short interfering nucleic acid (siNA)
EP3926046A2 (en) 2002-02-20 2021-12-22 Sirna Therapeutics, Inc. Rna interference mediated inhibition of gene expression using short interfering nucleic acid (sina)
US8013146B2 (en) 2002-02-20 2011-09-06 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of matrix metalloproteinase 13 (MMP13) gene expression using short interfering nucleic acid (siNA)
US8013143B2 (en) 2002-02-20 2011-09-06 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of CXCR4 gene expression using short interfering nucleic acid (siNA)
EP2042510A2 (en) 2002-02-20 2009-04-01 Sirna Therapeutics Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleid acid (siNA)
WO2003070966A2 (en) * 2002-02-20 2003-08-28 Sirna Therapeutics, Inc RNA INTERFERENCE MEDIATED TARGET DISCOVERY AND TARGET VALIDATION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US9732344B2 (en) 2002-02-20 2017-08-15 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10889815B2 (en) 2002-02-20 2021-01-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9738899B2 (en) 2002-02-20 2017-08-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US9771588B2 (en) 2002-02-20 2017-09-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US8067575B2 (en) 2002-02-20 2011-11-29 Merck, Sharp & Dohme Corp. RNA interference mediated inhibition of cyclin D1 gene expression using short interfering nucleic acid (siNA)
US8076472B2 (en) 2002-02-20 2011-12-13 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of muscarinic colinergic receptor gene expression using short interfering nucleic acid (siNA)
WO2003070918A2 (en) 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Rna interference by modified short interfering nucleic acid
US7985853B2 (en) 2002-02-20 2011-07-26 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of platelet derived growth factor (PDGF) and platelet derived growth factor receptor (PDGFR) gene expression using short interfering nucleic acid (siNA)
WO2003070972A2 (en) * 2002-02-20 2003-08-28 Sirna Therapeutics Inc. RNA INTERFERENCE MEDIATED INHIBITION OF CHROMOSOME TRANSLOCATION GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7795422B2 (en) 2002-02-20 2010-09-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hypoxia inducible factor 1 (HIF1) gene expression using short interfering nucleic acid (siNA)
US7700760B2 (en) 2002-02-20 2010-04-20 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular cell adhesion molecule (VCAM) gene expression using short interfering nucleic acid (siNA)
US7659390B2 (en) 2002-02-20 2010-02-09 Sirna Therapeutics, Inc. RNA interference mediated inhibition of muscarinic colinergic receptor gene expression using short interfering nucleic acid (siNA)
US7662952B2 (en) 2002-02-20 2010-02-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of GRB2 associated binding protein (GAB2) gene expression using short interfering nucleic acid (siNA)
EP3354656A1 (en) 2002-02-20 2018-08-01 Sirna Therapeutics, Inc. Rna interference mediated inhibition of gene expression using short interfering nucleic acid (sina)
US10351852B2 (en) 2002-02-20 2019-07-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10000754B2 (en) 2002-02-20 2018-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US7989612B2 (en) 2002-02-20 2011-08-02 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10662428B2 (en) 2002-02-20 2020-05-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US7667030B2 (en) 2002-02-20 2010-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of matrix metalloproteinase 13 (MMP13) gene expression using short interfering nucleic acid (siNA)
US7667029B2 (en) 2002-02-20 2010-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of checkpoint kinase-1 (CHK-1) gene expression using short interfering nucleic acid (siNA)
EP2278004A1 (en) 2002-02-20 2011-01-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US9957517B2 (en) 2002-02-20 2018-05-01 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
WO2003076620A1 (en) 2002-03-14 2003-09-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for monitoring and modulating gene silencing
US7601888B2 (en) 2002-03-21 2009-10-13 Monsanto Technology L.L.C. Nucleic acid constructs and methods for producing altered seed oil compositions
US7566813B2 (en) 2002-03-21 2009-07-28 Monsanto Technology, L.L.C. Nucleic acid constructs and methods for producing altered seed oil compositions
US8802922B2 (en) 2002-03-21 2014-08-12 Monsanto Technology Llc Nucleic acid constructs and methods for producing altered seed oil compositions
US10280430B2 (en) 2002-03-21 2019-05-07 Monsanto Technology Llc Nucleic acid constructs and methods for producing altered seed oil compositions
US8299042B2 (en) 2002-04-26 2012-10-30 Alnylam Pharmaceuticals, Inc. Methods and compositions for silencing genes without inducing toxicity
US9856476B2 (en) 2002-05-03 2018-01-02 Duke University Method of regulating gene expression
US9850485B2 (en) 2002-05-03 2017-12-26 Duke University Method of regulating gene expression
US9267145B2 (en) 2002-05-03 2016-02-23 Duke University Method of regulating gene expression
US10233451B2 (en) 2002-05-03 2019-03-19 Duke University Method of regulating gene expression
US7166771B2 (en) 2002-06-21 2007-01-23 Monsanto Technology Llc Coordinated decrease and increase of gene expression of more than one gene using transgenic constructs
US8481710B2 (en) 2002-08-05 2013-07-09 University Of Iowa Research Foundation RNA interference suppression of neurodegenerative diseases and methods of use thereof
US8779116B2 (en) 2002-08-05 2014-07-15 University Of Iowa Research Foundation SiRNA-mediated gene silencing
US9487779B2 (en) 2002-08-05 2016-11-08 University Of Iowa Research Foundation siRNA-mediated gene silencing
US9790501B2 (en) 2002-08-05 2017-10-17 Silence Therapeutics Gmbh Interfering RNA molecules
US8324370B2 (en) 2002-08-05 2012-12-04 Silence Therapeutics Aktiengesellschaft (Ag) Interfering RNA molecules
US10774332B2 (en) 2002-08-05 2020-09-15 Silence Therapeutics Gmbh Interfering RNA molecules
US8329890B2 (en) 2002-08-05 2012-12-11 University Of Iowa Research Foundation SiRNA-mediated gene silencing
US8933215B2 (en) 2002-08-05 2015-01-13 Silence Therapeutics Aktiengesellschaft (Ag) Interfering RNA molecules
US10329568B2 (en) 2002-08-05 2019-06-25 Silence Therapeutics Gmbh Interfering RNA molecules
US8524879B2 (en) 2002-08-05 2013-09-03 University Of Iowa Research Foundation RNA interference suppresion of neurodegenerative diseases and methods of use thereof
US10266829B2 (en) 2002-08-05 2019-04-23 Silence Therapeutics Gmbh Interfering RNA molecules
US9758784B1 (en) 2002-08-05 2017-09-12 Silence Therapeutics Gmbh Interfering RNA molecules
US7893245B2 (en) 2002-08-05 2011-02-22 Silence Therapeutics Aktiengesellschaft (Ag) Interfering RNA molecules
US7452987B2 (en) 2002-08-05 2008-11-18 Silence Therapeutics Aktiengesellschaft (Ag) Interfering RNA molecules
US10323246B2 (en) 2002-08-05 2019-06-18 Silence Therapeutics Gmbh Interfering RNA molecules
US9260716B2 (en) 2002-08-05 2016-02-16 University Of Iowa Research Foundation RNA interference suppression of neurodegenerative diseases and methods of use thereof
US9783802B2 (en) 2002-08-05 2017-10-10 Silence Therapeutics Gmbh Interfering RNA molecules
US10072264B2 (en) 2002-08-05 2018-09-11 University Of Iowa Research Foundation RNA interference suppression of neurodegenerative diseases and methods of use
US11578328B2 (en) 2002-08-05 2023-02-14 Silence Therapeutics Gmbh Interfering RNA molecules
US9695423B2 (en) 2002-08-05 2017-07-04 Silence Therapeutics Gmbh Interfering RNA molecules
US9790505B2 (en) 2002-08-05 2017-10-17 Silence Therapeutics Gmbh Interfering RNA molecules
US9222092B2 (en) 2002-08-05 2015-12-29 Silence Therapeutics Gmbh Interfering RNA molecules
US10130737B2 (en) 2002-08-21 2018-11-20 Revivicor, Inc. Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US11172658B2 (en) 2002-08-21 2021-11-16 Revivicor, Inc. Porcine animals lacking expression of functional alpha 1, 3 galactosyltransferase
US7795493B2 (en) 2002-08-21 2010-09-14 Revivicor, Inc. Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US8106251B2 (en) 2002-08-21 2012-01-31 Revivicor, Inc. Tissue products derived from porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase
US10912863B2 (en) 2002-08-21 2021-02-09 Revivicor, Inc. Tissue products derived from animals lacking any expression of functional alpha 1, 3 galactosyltransferase
US7956176B2 (en) 2002-09-05 2011-06-07 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US7923547B2 (en) 2002-09-05 2011-04-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
WO2004041838A1 (en) * 2002-11-01 2004-05-21 University Of Massachusetts Regulation of transcription elongation factors
EP1445321A1 (en) 2002-12-18 2004-08-11 Monsanto Technology LLC Maize embryo-specific promoter compositions and methods for use thereof
EP2116606A1 (en) 2003-03-28 2009-11-11 Monsanto Technology, LLC Novel plant promoters for use in early seed development
EP2116607A1 (en) 2003-03-28 2009-11-11 Monsanto Technology, LLC Novel plant promoters for use in early seed development
US7960614B2 (en) 2003-06-06 2011-06-14 Arborgen, Llc Plant transformation and selection
US8633028B2 (en) * 2003-07-02 2014-01-21 Musc Foundation For Research Development dsRNA induced specific and non-specific immunity in crustaceans and other invertebrates and biodelivery vehicles for use therein
US7695426B2 (en) 2003-08-20 2010-04-13 Biological Resources Pty Ltd Methods for enhancing viability
US8716554B2 (en) 2003-08-21 2014-05-06 Rahan Meristem (1998) Ltd. Plant Propagation & Biotechnology Plants resistant to cytoplasm-feeding parasites
US7795504B2 (en) 2003-09-24 2010-09-14 Monsanto Technology Llc Coordinated decrease and increase of gene expression of more than one gene using transgenic constructs
US7560538B2 (en) 2003-11-05 2009-07-14 University Of Pittsburgh Porcine isogloboside 3 synthase protein, cDNA, genomic organization, and regulatory region
WO2005081714A2 (en) 2003-11-21 2005-09-09 Revivicor, Inc. Use of interfering rna in the production of transgenic animals
US10793873B2 (en) 2003-11-21 2020-10-06 Revivicor, Inc. Use of interfering RNA in the production of transgenic animals
US9534252B2 (en) 2003-12-01 2017-01-03 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
WO2005060739A1 (en) 2003-12-24 2005-07-07 G2 Inflammation Pty Ltd Transgenic non-human mammal comprising a polynucleotide encoding human or humanized c5ar
EP2322637A1 (en) 2003-12-24 2011-05-18 G2 Inflammation Pty Ltd Transgenic non-human mammal comprising a polynucleotide encoding human or humanized C5AR
US7683237B2 (en) 2004-02-10 2010-03-23 Monsanto Technology Llc Maize seed with synergistically enhanced lysine content
US7858769B2 (en) 2004-02-10 2010-12-28 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using multifunctional short interfering nucleic acid (multifunctional siNA)
EP3290516A1 (en) 2004-02-10 2018-03-07 Monsanto Technology LLC Recombinant dna for gene suppression
US7855323B2 (en) 2004-02-10 2010-12-21 Monsanto Technology Llc Recombinant DNA for gene suppression
US9913487B2 (en) 2004-02-10 2018-03-13 Monsanto Technology Llc Enhanced zein reduction in transgenic corn seed
WO2005077116A2 (en) 2004-02-10 2005-08-25 Mosanto Technology, Llc Recombinant dna for gene suppression
US9006414B2 (en) 2004-02-10 2015-04-14 Monsanto Technology Llc Recombinant DNA for gene suppression
EP2365072A1 (en) 2004-02-10 2011-09-14 Monsanto Technology LLC Recombinant dna for gene suppression
US9976139B2 (en) 2004-02-10 2018-05-22 Monsanto Technology Llc Recombinant DNA for gene suppression
US10772276B2 (en) 2004-02-10 2020-09-15 Monsanto Technology Llc Enhanced zein reduction in transgenic corn seed
US8283461B2 (en) 2004-03-05 2012-10-09 Benitec, Inc. Multiple promoter expression cassettes for simultaneous delivery of RNAi agents
WO2005087926A3 (en) * 2004-03-05 2006-03-16 Benitec Inc Multiple promoter expression cassettes for simultaneous delivery of rnai agents
US7727970B2 (en) 2004-03-05 2010-06-01 Benitec, Inc. Multiple promoter expression cassettes for simultaneous delivery of RNAi agents targeted to Hepatitis C virus
US8691967B2 (en) 2004-03-05 2014-04-08 Benitec, Inc. Multiple promoter expression cassettes for simultaneous delivery of RNAi agents
EP2292773A1 (en) 2004-03-25 2011-03-09 Monsanto Technology LLC Genes and uses for plant improvement
US8049069B2 (en) 2004-03-31 2011-11-01 Commonwealth Scientific And Industrial Research Organisation Genes involved in plant fibre development
US8946510B2 (en) 2004-04-09 2015-02-03 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
EP2308971A1 (en) 2004-04-09 2011-04-13 Monsanto Technology LLC Compositions and methods for control of insect infestations in plants
US10167484B2 (en) 2004-04-09 2019-01-01 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US11492638B2 (en) 2004-04-09 2022-11-08 Monsanto Technology, Llc Compositions and methods for control of insect infestations in plants
WO2005110068A2 (en) 2004-04-09 2005-11-24 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
EP2402441A1 (en) 2004-04-09 2012-01-04 Monsanto Technology, LLC Compositions and methods for control of insect infestations in plants
US9238822B2 (en) 2004-04-09 2016-01-19 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US11685930B2 (en) 2004-04-09 2023-06-27 Monsanto Technology, Llc Compositions and methods for control of insect infestations in plants
US10787680B2 (en) 2004-04-09 2020-09-29 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US9340797B2 (en) 2004-04-09 2016-05-17 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
EP2357243A2 (en) 2004-04-22 2011-08-17 Commonwealth Scientific and Industrial Research Organisation Synthesis of long-chain polyunsaturated fatty acids by recombinant cells.
EP2357244A2 (en) 2004-04-22 2011-08-17 Commonwealth Scientific and Industrial Research Organisation Synthesis of long-chain polyunsaturated fatty acids by recombinant cells.
EP2363492A2 (en) 2004-04-22 2011-09-07 Commonwealth Scientific and Industrial Research Organisation Synthesis of long-chain polyunsaturated fatty acids by recombinant cells.
US8946402B2 (en) 2004-04-23 2015-02-03 The Trustees Of Columbia University In The City Of New York Inhibition of hairless protein mRNA
US8329667B2 (en) 2004-04-23 2012-12-11 The Trustees Of Columbia University In The City Of New York Inhibition of hairless protein mRNA
US10508277B2 (en) 2004-05-24 2019-12-17 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
EP1621632A1 (en) 2004-07-31 2006-02-01 Monsanto Technology, LLC Genes and uses for plant improvement
EP2295582A2 (en) 2004-07-31 2011-03-16 Monsanto Technology LLC Genes and uses for plant improvement
US8461418B2 (en) 2004-08-11 2013-06-11 Monsanto Technology Llc Enhanced zein reduction in transgenic corn seed
EP2330203A2 (en) 2004-10-25 2011-06-08 Devgen NV Rna constructs
EP2330203A3 (en) * 2004-10-25 2011-10-05 Devgen NV Rna constructs
WO2006046148A3 (en) * 2004-10-25 2006-11-30 Devgen Nv Rna constructs
WO2006046148A2 (en) 2004-10-25 2006-05-04 Devgen Nv Rna constructs
EP2003205A2 (en) 2004-12-28 2008-12-17 Pioneer Hi-Bred International, Inc. Improved grain quality through altered expression of seed proteins
EP2489726A2 (en) 2005-01-12 2012-08-22 Monsanto Technology LLC Genes and uses for plant improvement
EP2584033A1 (en) 2005-01-12 2013-04-24 Monsanto Technology LLC Genes and uses for plant improvement
EP3059306A1 (en) 2005-01-12 2016-08-24 Monsanto Technology LLC Genes and uses for plant improvement
EP2338905A2 (en) 2005-02-23 2011-06-29 North Carolina State University Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes
WO2006099249A2 (en) 2005-03-10 2006-09-21 Monsanto Technology Llc Maize seed with synergistically enhanced lysine content
WO2006133983A1 (en) 2005-04-19 2006-12-21 Basf Plant Science Gmbh Starchy-endosperm and/or germinating embryo-specific expression in mono-cotyledonous plants
EP2261362A2 (en) 2005-05-25 2010-12-15 Pioneer Hi-Bred International Inc. Methods for improving crop plant architecture and yield
EP2261361A2 (en) 2005-05-25 2010-12-15 Pioneer Hi-Bred International Inc. Methods for improving crop plant architecture and yield
EP2275563A2 (en) 2005-09-16 2011-01-19 deVGen N.V. Transgenic plant-based methods for plant insect pests using RNAi
US7943819B2 (en) 2005-09-16 2011-05-17 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
US10538783B2 (en) 2005-09-16 2020-01-21 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
EP2275562A2 (en) 2005-09-16 2011-01-19 deVGen N.V. Transgenic plant-based methods for plant insect pests using RNAi
EP3508582A1 (en) 2005-09-16 2019-07-10 Monsanto Technology LLC Methods for genetic control of insect infestations in plants and compositions thereof
EP2439278A1 (en) 2005-09-16 2012-04-11 Monsanto Technology LLC Methods for genetic control of insect infestations in plants and compositions thereof
EP2439279A1 (en) 2005-09-16 2012-04-11 Monsanto Technology LLC Methods for genetic control of insect infestations in plants and compositions thereof
EP2431473A1 (en) 2005-09-16 2012-03-21 Monsanto Technology LLC Methods for genetic control of insect infestations in plants and compositions thereof
EP3173486A1 (en) 2005-09-16 2017-05-31 Monsanto Technology LLC Methods for genetic control of insect infestations in plants and compositions thereof
US11312975B2 (en) 2005-09-16 2022-04-26 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
EP2426208A1 (en) 2005-09-16 2012-03-07 Monsanto Technology, LLC Methods for genetic control of insect infestations in plants and compositions thereof
US9695439B2 (en) 2005-09-16 2017-07-04 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
EP2330207A2 (en) 2005-09-16 2011-06-08 deVGen N.V. Transgenic plant-based methods for plant pests using RNAi
US8759611B2 (en) 2005-09-16 2014-06-24 Monsanto Technology Llc Methods for genetic control of insect infestation in plants and compositions thereof
EP2281896A2 (en) 2005-09-16 2011-02-09 deVGen N.V. Transgenic plant-based methods for plant insect pests using RNAi
EP2295584A2 (en) 2005-09-16 2011-03-16 deVGen N.V. Transgenic plant-based methods for plant pests using RNAi
EP2980220A1 (en) 2005-09-20 2016-02-03 BASF Plant Science GmbH Improved methods controlling gene expression
US8093369B2 (en) 2005-10-11 2012-01-10 Ben Gurion University Of The Negev Research And Development Authority Ltd. Compositions for silencing the expression of VDAC1 and uses thereof
US8269082B2 (en) 2005-10-20 2012-09-18 Commonwealth Scientific And Industrial Research Organisation Cereals with altered dormancy
US7884264B2 (en) 2006-01-17 2011-02-08 Biolex Therapeutics, Inc. Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in duckweed plants
US8716557B2 (en) 2006-01-17 2014-05-06 Synthon Biopharmaceuticals B.V. Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in plants
EP2730587A2 (en) 2006-02-09 2014-05-14 Pioneer Hi-Bred International, Inc. Genes for enhancing nitrogen utilization efficiency in crop plants
WO2007095469A2 (en) 2006-02-10 2007-08-23 Monsanto Technology Llc Identification and use of target genes for control of plant parasitic nematodes
US9765351B2 (en) 2006-02-13 2017-09-19 Monsanto Technology Llc Modified gene silencing
US10941398B2 (en) 2006-02-13 2021-03-09 Monsanto Technology Llc Selecting and stabilizing dsRNA constructs
EP2426206A2 (en) 2006-02-13 2012-03-07 Monsanto Technology LLC Selecting and stablizing dsRNA constructs
US11708577B2 (en) 2006-02-13 2023-07-25 Monsanto Technology Llc Modified gene silencing
EP2292739A1 (en) 2006-03-24 2011-03-09 Institut National De La Recherche Agronomique Method for preparing differentiated avian cells and genes involved in the maintenance of pluripotency
EP2251349A1 (en) 2006-04-19 2010-11-17 Pioneer Hi-Bred International, Inc. Isolated polynucleotide molecules corresponding to mutant and wild-type alleles of the maize D9 gene and methods of use
US8536429B2 (en) 2006-07-12 2013-09-17 Commonwealth Scientific And Industrial Research Organisation Polynucleotides encoding a NAX2 polypeptide and methods for enhancing salinity tolerance in plants
US10301623B2 (en) 2006-08-31 2019-05-28 Monsanto Technology Llc Phased small RNAs
US9309512B2 (en) 2006-08-31 2016-04-12 Monsanto Technology Llc Phased small RNAs
EP2059596B1 (en) * 2006-08-31 2017-10-04 Monsanto Technology, LLC Phased small rnas
US8895805B2 (en) 2006-12-04 2014-11-25 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences Method for modifying insect resistance of plants by utilizing RNAi technique
US9290777B2 (en) 2007-02-05 2016-03-22 National University Of Singapore Putative cytokinin receptor and methods for use thereof
US8796442B2 (en) 2007-03-21 2014-08-05 Brookhaven Science Associates, Llc. Combined hairpin-antisense compositions and methods for modulating expression
US9193972B2 (en) 2007-03-21 2015-11-24 Brookhaven Science Associates, Llc Combined hairpin-antisense compositions and methods for modulating expression
WO2008116094A2 (en) 2007-03-21 2008-09-25 Brookhaven Science Associates, Llc Combined hairpin-antisense compositions and methods for modulating expression
US10201601B2 (en) 2007-03-26 2019-02-12 University Of Massachusetts Compositions and methods for increasing immunogenicity of glycoprotein vaccines
US20100145015A1 (en) * 2007-03-26 2010-06-10 Uri Galili Compositions and methods for increasing immunogenicity of glycoprotein vaccines
US9662383B2 (en) * 2007-03-26 2017-05-30 University Of Massachusetts Compositions and methods for increasing immunogenicity of glycoprotein vaccines
WO2008157263A2 (en) 2007-06-15 2008-12-24 Arkansas State University Methods of delivery of molecules to cells using a ricin subunit and compositions relating to same
US8642846B2 (en) 2007-08-13 2014-02-04 Commonwealth Scientific And Industrial Research Organisation Barley with low levels of hordeins
US11326134B2 (en) 2007-08-13 2022-05-10 Commonwealth Scientific And Industrial Research Organisation Barley with low levels of hordeins
US9133427B2 (en) 2007-08-13 2015-09-15 Commonwealth Scientific And Industrial Research Organisation Barley with low levels of hordeins
WO2009021285A1 (en) 2007-08-13 2009-02-19 Commonwealth Scientific And Industrial Research Organisation Barley with low levels of hordein
US10501712B2 (en) 2007-08-13 2019-12-10 Commonwealth Scientific And Industrial Research Organisation Barley with low levels of hordeins
US9885038B2 (en) 2007-08-14 2018-02-06 Commonwealth Scientific & Industrial Research Organisation Gene silencing methods
WO2009026660A1 (en) 2007-08-30 2009-03-05 Walter And Eliza Hall Institute Of Medical Research Dendritic cell marker and uses thereof
WO2009067580A2 (en) 2007-11-20 2009-05-28 Pioneer Hi-Bred International, Inc. Maize ethylene signaling genes and modulation of same for improved stress tolerance in plants
WO2009067751A1 (en) 2007-11-27 2009-06-04 Commonwealth Scientific And Industrial Research Organisation Plants with modified starch metabolism
WO2009129558A1 (en) 2008-04-24 2009-10-29 Newsouth Innovations Pty Limited Cyanobacteria saxitoxin gene cluster and detection of cyanotoxic organisms
US8093043B2 (en) 2008-06-04 2012-01-10 New York University β-TrCP1, β-TrCP2 and RSK1 or RSK2 inhibitors and methods for sensitizing target cells to apoptosis
WO2010005527A1 (en) 2008-06-30 2010-01-14 Angioblast Systems, Inc. Treatment of eye diseases and excessive neovascularization using a combined therapy
WO2010009499A1 (en) 2008-07-21 2010-01-28 Commonwealth Scientific And Industrial Research Organisation Improved cottonseed oil and uses
US8692080B2 (en) 2008-09-29 2014-04-08 Monsanto Technology Llc Soybean transgenic event MON87705 and methods for detection thereof
US9572311B2 (en) 2008-09-29 2017-02-21 Monsanto Technology Llc Soybean transgenic event MON87705 and methods for detection thereof
US10344292B2 (en) 2008-09-29 2019-07-09 Monsanto Technology Llc Soybean transgenic event MON87705 and methods for detection thereof
US8329989B2 (en) 2008-09-29 2012-12-11 Monsanto Technology Llc Soybean transgenic event MON87705 and methods for detection thereof
EP2821490A2 (en) 2008-10-30 2015-01-07 Pioneer Hi-Bred International Inc. Manipulation of glutamine synthetases (GS) to improve nitrogen use efficiency and grain yield in higher plants
WO2010065867A1 (en) 2008-12-04 2010-06-10 Pioneer Hi-Bred International, Inc. Methods and compositions for enhanced yield by targeted expression of knotted1
WO2010066689A2 (en) 2008-12-09 2010-06-17 Novartis Ag Organic compounds
WO2010101818A1 (en) 2009-03-02 2010-09-10 Pioneer Hi-Bred International, Inc. Nac transcriptional activators involved in abiotic stress tolerance
EP3293257A1 (en) 2009-03-20 2018-03-14 Mesoblast, Inc. Production of reprogrammed pluripotent cells
WO2010120862A1 (en) 2009-04-14 2010-10-21 Pioneer Hi-Bred International, Inc. Modulation of acc synthase improves plant yield under low nitrogen conditions
WO2010118477A1 (en) 2009-04-17 2010-10-21 Molecular Plant Breeding Nominees Ltd Plant promoter operable in endosperm and uses thereof
EP3260545A1 (en) 2009-04-20 2017-12-27 Monsanto Technology LLC Multiple virus resistance in plants
WO2010123904A1 (en) 2009-04-20 2010-10-28 Monsanto Technology Llc Multiple virus resistance in plants
WO2010139026A1 (en) 2009-06-05 2010-12-09 Centenary Institute Of Cancer Medicine And Cell Biology Therapeutic and diagnostic molecules
WO2011011273A1 (en) 2009-07-24 2011-01-27 Pioneer Hi-Bred International, Inc. The use of dimerization domain component stacks to modulate plant architecture
WO2011041796A1 (en) 2009-10-02 2011-04-07 Pioneer Hi-Bred International, Inc. Down-regulation of acc synthase for improved plant performance
EP3766976A1 (en) 2009-12-18 2021-01-20 Arrowhead Pharmaceuticals, Inc. Organic compositions to treat hsf1-related diseases
EP3000885A2 (en) 2009-12-18 2016-03-30 Arrowhead Research Corporation Organic compositions to treat hsf1-related diseases
WO2011073326A2 (en) 2009-12-18 2011-06-23 Novartis Ag Organic compositions to treat hsf1-related diseases
EP3406720A1 (en) 2009-12-18 2018-11-28 Arrowhead Pharmaceuticals, Inc. Organic compositions to treat hsf1-related diseases
WO2011085062A1 (en) 2010-01-06 2011-07-14 Pioneer Hi-Bred International, Inc. Identification of diurnal rhythms in photosynthetic and non-photosynthetic tissues from zea mays and use in improving crop plants
WO2011098449A1 (en) 2010-02-10 2011-08-18 Novartis Ag Methods and compounds for muscle growth
WO2012007945A2 (en) 2010-07-12 2012-01-19 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization, (A.R.O.), Volcani Center Isolated polynucleotides and methods and plants using same for regulating plant acidity
EP3124610A1 (en) 2010-10-28 2017-02-01 Benitec Biopharma Limited Hbv treatment
WO2012055362A1 (en) 2010-10-28 2012-05-03 Benitec Biopharma Limited Hbv treatment
US11193126B2 (en) 2010-10-29 2021-12-07 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
US9260471B2 (en) 2010-10-29 2016-02-16 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
US9970005B2 (en) 2010-10-29 2018-05-15 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)
WO2012058730A1 (en) 2010-11-04 2012-05-10 Arista Cereal Technologies Pty Ltd High amylose wheat
WO2012058726A1 (en) 2010-11-05 2012-05-10 Transbio Ltd Markers of endothelial progenitor cells and uses thereof
WO2012112586A1 (en) 2011-02-14 2012-08-23 Revivicor, Inc. Genetically modified pigs for xenotransplantation of vascularized xenografts and derivatives thereof
US11179496B2 (en) 2011-02-14 2021-11-23 Revivicor, Inc. Genetically modified pigs for xenotransplantation of vascularized xenografts and derivatives thereof
WO2012129373A2 (en) 2011-03-23 2012-09-27 Pioneer Hi-Bred International, Inc. Methods for producing a complex transgenic trait locus
WO2012148835A1 (en) 2011-04-29 2012-11-01 Pioneer Hi-Bred International, Inc. Down-regulation of a homeodomain-leucine zipper i-class homeobox gene for improved plant performance
WO2012174139A2 (en) 2011-06-14 2012-12-20 Synthon Biopharmaceuticals B.V. Compositions and methods for making and b ioc ont aining auxotrophic transgenic plants
WO2013066423A2 (en) 2011-06-21 2013-05-10 Pioneer Hi-Bred International, Inc. Methods and compositions for producing male sterile plants
WO2013066805A1 (en) 2011-10-31 2013-05-10 Pioneer Hi-Bred International, Inc. Improving plant drought tolerance, nitrogen use efficiency and yield
WO2013063653A1 (en) 2011-11-04 2013-05-10 Arista Cereal Technologies Pty Limited High amylose wheat - ii
WO2013088438A1 (en) 2011-12-11 2013-06-20 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization, (A.R.O.), Volcani Center Methods of modulating stomata conductance and plant expression constructs for executing same
WO2013096992A1 (en) 2011-12-27 2013-07-04 Commonwealth Scientific And Industrial Reserach Organisation Simultaneous gene silencing and supressing gene silencing in the same cell
US10822615B2 (en) 2011-12-27 2020-11-03 Commonwealth Scientific And Industrial Research Organisation Simultaneous gene silencing and suppressing gene silencing in the same cell
WO2013096993A1 (en) 2011-12-27 2013-07-04 Commonwealth Scientific And Industrial Research Organisation Processes for producing lipids
US11560571B2 (en) 2011-12-27 2023-01-24 Commonwealth Scientific And Industrial Research Organisation Simultaneous gene silencing and suppressing gene silencing in ihe same cell
WO2013096991A1 (en) 2011-12-27 2013-07-04 Commonwealth Scientific And Industrial Research Organisation Production of dihydrosterculic acid and derivatives thereof
WO2013105022A2 (en) 2012-01-09 2013-07-18 Novartis Ag Organic compositions to treat beta-catenin-related diseases
WO2013104026A1 (en) 2012-01-11 2013-07-18 The Australian National University Method for modulating plant root architecture
WO2013138358A1 (en) 2012-03-13 2013-09-19 Pioneer Hi-Bred International, Inc. Genetic reduction of male fertility in plants
WO2013138309A1 (en) 2012-03-13 2013-09-19 Pioneer Hi-Bred International, Inc. Genetic reduction of male fertility in plants
WO2013159149A1 (en) 2012-04-25 2013-10-31 Commonwealth Scientific And Industrial Research Organisation High oleic acid oils
WO2014027021A1 (en) 2012-08-16 2014-02-20 Vib Vzw Means and methods for altering the lignin pathway in plants
US10006041B2 (en) 2012-08-16 2018-06-26 Vib Vzw Means and methods for altering the lignin pathway in plants
WO2014118123A1 (en) 2013-01-29 2014-08-07 The University Court Of The University Of Glasgow Methods and means for increasing stress tolerance and biomass in plants
WO2014164014A1 (en) 2013-03-11 2014-10-09 Pioneer Hi-Bred International, Inc. Genes for improving nutrient uptake and abiotic stress tolerance in plants
WO2014164116A1 (en) 2013-03-13 2014-10-09 Pioneer Hi-Bred International, Inc. Functional expression of bacterial major facilitator superfamily (sfm) gene in maize to improve agronomic traits and grain yield
WO2014164074A1 (en) 2013-03-13 2014-10-09 Pioneer Hi-Bred International, Inc. Enhanced nitrate uptake and nitrate translocation by over-expressing maize functional low-affinity nitrate transporters in transgenic maize
WO2014160122A1 (en) 2013-03-14 2014-10-02 Pioneer Hi-Bred International, Inc. Maize stress related transcription factor 18 and uses thereof
WO2014143996A2 (en) 2013-03-15 2014-09-18 Pioneer Hi-Bred International, Inc. Compositions and methods of use of acc oxidase polynucleotides and polypeptides
US10017778B2 (en) 2013-03-21 2018-07-10 Vib Vzw Means and methods for the reduction of photorespiration in crops
WO2014147249A1 (en) 2013-03-21 2014-09-25 Vib Vzw Means and methods for the reduction of photorespiration in crops
EP2810952A1 (en) 2013-06-03 2014-12-10 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Novel pest control methods
WO2014200842A2 (en) 2013-06-11 2014-12-18 Syngenta Participations Ag Methods for generating transgenic plants
US10849345B2 (en) 2013-06-13 2020-12-01 Commonwealth Scientific And Industrial Research Organisation Barley with very low levels of hordeins
EP3682873A1 (en) 2013-06-25 2020-07-22 The Walter and Eliza Hall Institute of Medical Research Smac mimetics for use in the treatment of persistent hiv infection
US10500252B2 (en) 2013-06-25 2019-12-10 Walter And Eliza Hall Institute Of Medical Research Method of treating intracellular infection
WO2014205516A1 (en) 2013-06-25 2014-12-31 The Walter And Eliza Hall Institute Of Medical Research Method of treating intracellular infection
US10428336B2 (en) 2013-10-16 2019-10-01 The Australian National University Method for modulating plant growth
WO2015069459A1 (en) 2013-11-05 2015-05-14 Novartis Ag Organic compounds
US11261243B2 (en) 2013-11-28 2022-03-01 Csl Limited Methods of treating diabetic nephropathy by administering an anti-vegf-b (vascular endothelial growth factor-b) antibody
US10407498B2 (en) 2013-11-28 2019-09-10 Csl Limited Method of treating diabetic nephropathy in a subject suffering from type 2 diabetes by administering an antibody which inhibits VEGF-B signaling
US9803008B2 (en) 2013-11-28 2017-10-31 Csl Limited Method of treating diabetic nephropathy by administering antibodies to vascular endothelial growth factor B (VEGF-B)
WO2015089585A1 (en) 2013-12-18 2015-06-25 Csl Limited Method of treating wounds
WO2015116680A1 (en) 2014-01-30 2015-08-06 Two Blades Foundation Plants with enhanced resistance to phytophthora
WO2015171603A1 (en) 2014-05-06 2015-11-12 Two Blades Foundation Methods for producing plants with enhanced resistance to oomycete pathogens
EP4303288A2 (en) 2014-07-07 2024-01-10 Nuseed Global Innovation Ltd Processes for producing industrial products from plant lipids
EP2966157A1 (en) 2014-07-07 2016-01-13 Commonwealth Scientific and Industrial Research Organisation Processes for producing industrial products from plant lipids
WO2016005449A1 (en) 2014-07-08 2016-01-14 Vib Vzw Means and methods to increase plant yield
WO2016079527A1 (en) 2014-11-19 2016-05-26 Tetralogic Birinapant Uk Ltd Combination therapy
WO2016097773A1 (en) 2014-12-19 2016-06-23 Children's Cancer Institute Therapeutic iap antagonists for treating proliferative disorders
WO2016196489A1 (en) 2015-05-29 2016-12-08 Arcadia Biosciences Reduced gluten grains and compositions thereof
EP4282875A2 (en) 2015-05-29 2023-11-29 Arcadia Biosciences, Inc. Reduced gluten grains and compositions thereof
WO2017062790A1 (en) 2015-10-09 2017-04-13 Two Blades Foundation Cold shock protein receptors and methods of use
WO2017083920A1 (en) 2015-11-18 2017-05-26 Commonwealth Scientific And Industrial Research Organisation Rice grain with thickened aleurone
WO2017091952A1 (en) 2015-11-30 2017-06-08 谢彦晖 Use of akt2 in diagnosis and treatment of tumor
WO2017161264A1 (en) 2016-03-18 2017-09-21 Pioneer Hi-Bred International, Inc. Methods and compositions for producing clonal, non-reduced, non-recombined gametes
CN107841510B (en) * 2016-09-20 2021-02-09 中国科学院青岛生物能源与过程研究所 Method for controlling expression ratio of different genes horizontally after transcription of prokaryotic cell
CN107841510A (en) * 2016-09-20 2018-03-27 中国科学院青岛生物能源与过程研究所 A kind of method of prokaryotic post-transcriptional level control different genes expression ratio
WO2020078865A1 (en) 2018-10-16 2020-04-23 F. Hoffmann-La Roche Ag Use of akt inhibitors in ophthalmology
WO2020148310A1 (en) 2019-01-17 2020-07-23 F. Hoffmann-La Roche Ag E3 ubiquitin ligase (ube3a) protein targets
WO2021019536A1 (en) 2019-07-30 2021-02-04 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Methods of controlling cannabinoid synthesis in plants or cells and plants and cells produced thereby
EP3825408A1 (en) 2019-11-19 2021-05-26 FRAUNHOFER-GESELLSCHAFT zur Förderung der angewandten Forschung e.V. Methods of multi-species insect pest control
WO2021099377A1 (en) 2019-11-19 2021-05-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Methods of multi-species insect pest control
WO2022162015A1 (en) 2021-01-26 2022-08-04 Universite Brest Bretagne Occidentale Novel stim1 splicing variants and uses thereof
WO2023012342A1 (en) 2021-08-06 2023-02-09 Kws Vegetables B.V. Durable downy mildew resistance in spinach
WO2023020980A1 (en) 2021-08-16 2023-02-23 F. Hoffmann-La Roche Ag E3 ubiquitin ligase (ube3a) protein targets
US11932854B2 (en) 2021-10-25 2024-03-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)

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