WO1986006409A1

WO1986006409A1 - High level amplification and expression of exogenous dna

Info

Publication number: WO1986006409A1
Application number: PCT/US1986/000934
Authority: WO
Inventors: Randal J. Kaufman
Original assignee: Genetics Institute, Inc.
Priority date: 1985-05-01
Filing date: 1986-04-28
Publication date: 1986-11-06
Also published as: EP0221955A4; EP0221955A1; JPS62502662A

Abstract

A method for producing high level expression of a selected protein and cell line and vectores useful therein. This method involves incorporating an exogenous ADA gene and an exogenous gene coding for a desired protein into a cell line containing an endogenous ADA gene.

Description

HIGH LEVEL AMPLIFICATION AND EXPRESSION OF EXOGENOUS DNA

Background

This invention relates to amethod andunique expression vectors that use heterologous adenosine dea inase (ADA) DNA as a selectable marker for transformation and/or as a co- amplifier of DNA coding for an exogenous protein in a host cell containing endogenous ADA.

Transformation is a commonly-employed genetic engineer- ing procedure in which new genetic material is acquired by eukaryotic or procaryotic cells by the incorporation of exogenous DNA sequences coding for a desired protein, polypeptide, and the like. Ordinarily, the number of cells in a population undergoing transformation which actually incorporate the exogenous DNA is quite low.

These problems can be obviated by transforming the cell with a selection marker in addition to the exogenous DNA sequence. Depending upon whether and how closely the selection marker is linked to the exogenous protein-encoding DNA, cells carrying the selection marker will also contain the exogenous DNA. Using appropriate conditions, cells transformed with the selection marker can be distinguished from cells that have not incorporated the exogenous DNA. Selection involves the use of DNA encoding an easily- identifiable marker, for example, resistance to an anti¬ biotic. Upon transformation, the cell population is examined for the presence of the marker. Those cells which have successfully incorporated the marker DNA will exhibit the marker identity (e.g. survival in media containing the antibiotic) and those cells which have failed to incorporate the marker will not exhibit the marker feature (e.g. they will die .upon exposure to the antibiotic) .

The level of exogenous protein expressed by the transformed cells can be substantially increased where a DNA encoding an amplifiable gene as well as a selectable marker is included in the transformation process. Amplifi¬ cation of a gene involves exposing the transformed cell to environmental pressure sufficient to require the cell to produce more copies of the amplifiable gene for survival. Accordingly, the use of gene amplification for the high level expression of exogenous genes is an important technique.

The marker/amplification system most extensively used employs the gene for dihydrofolate reductase (DHFR) , a fairly ubiquitous gene found in many cell lines. Exposing a cell transformed with DHFR-encoding DNA to cytotoxic concen¬ trations of methotrexate (MTX) encourages the cell to amplify DHFR to survive. Cells which survive the MTX selection procedure have many copies of the DNA encoding DHFR. When the DHFR gene is on a plasmid containing a DNA sequence for another gene, that gene generally becomes amplified as well. Thus when transforming a cell with a vector containing a DHFR gene and an exogenous gene, the DHFR behaves as a selectable marker to enable the identifi¬ cation of those cells which have incorporated the vector from those cells which have not and also is capable of being itself amplified and consequently amplifying the exogenous DNA. The use of the DHFR gene both as a selectable and amplifiable marker has become widespread for deriving transformed cell lines. However, in practice, the DHFR system has demonstrated general utility only with one cell line, a Chinese hamster ovary line which is deficient in DHFR (CHO DHFR") . [Urlaub et al, Proc. Natl. Acad. Sci. U.S.A.. 72:4216-4220 (1982)]. Cell lines containing endogenous DHFRgenes cannot be employed because the endogenous DHFR prevents selection of those cells containing the DHFR and exogenous gene-containing vector. A mutant DHFR gene has been reported which purportedly can be expressed when inserted into cell lines containing endogenous DHFR. [Si onson, C.C. et al., Proc. Natl. Acad. Sci. U.S.A.. 80: 2495-99, (1983)]. However, these cell lines cannot be significantly amplified and are of marginal utility in attempting to obtain the high level of exogenous polypeptide desired from transformed cells. The construction of a selectable marker enabling the use of DHFR in cell lines possessing the DHFR gene has been reported by Murray,

M.J. et al., Mol. Cell. Biol. 2: 32-43 (1983). However, obtaining the optimal conditions necessary for expression of exogenous proteins in such cell lines has proven difficult.

Thus, expression and amplification of exogenous protein with the DHFR system has been limited to a single cell line, which is not always the cell line of choice for producing the desired protein. Other cell lines produce specific proteins at a greater level than, or will grow better than, CHO DHFR" under specified conditions. Other systems for amplifying and expressing heterologous DNA in a variety of different cell lines remain an unfulfilled need in the art. SummaryOf the Invention

As one aspect of the present invention, it is sur- prisingly discovered that an exogenous adenosine dea inase (ADA) gene may be used as a selectable and amplifiable marker in cell lines containing an endogenous ADA gene. A gene encoding ADA is present in virtually all mammalian tissues, but is not an essential enzyme for cell growth. [See Shipman, C. Jr., et al.. Science 2001 1163-1165 (1978); Hirschorn, R. et al., Proc. Natl. Acad. Sci. U.S.A. 73; 213-217 (1976)]. The method of the present invention thus makes possible the amplification of exogenous DNA coding for a desired protein in a wide variety of ADA+ eucaryotic cells, particularly mammalian cells. This method involves incorporating an exogenous ADA gene and a heterologous gene coding for a desired protein into a cell line containing an endogenous ADA gene. Cells containing the exogenous ADA gene and the heterologous protein gene are then selected and the genes amplified. Finally, the heterologous protein gene is expressed and the desired protein recovered.

As another aspect of the present invention, a cell line is provided for use in the ADA amplification method. The cell line is produced by transforming a cell containing endogenous ADA with an exogenous gene coding for ADA and an exogenous gene coding for the desired protein and coamplifying these exogenous genes. The resulting cell line with its amplified ADA and protein genes may then be cultured according to the present invention. High levels of the desired protein are expressed thereby. The ADA gene so employed can be the presently known sequence, of either human ADA or urine ADA. Depending on the use to which the protein is to be put, however, other species ADA genes may be used in analogous fashion. As a further aspect of the present invention, novel vectors are provided which incorporate exogenous ADA genes and exogenous genes coding for a desired protein. These vectors contain polyoma or retroviral sequences and can be employed to transform ADA⁺ cells or cell lines for use in the method of the invention to produce the desired protein.

Unlike the DHFR amplification system which requires use of a DHFR" cell line, the ADA amplification method makes possible the employment of many ADA+ cells and ADA⁴" cell lines that will grow best under specific conditions and/or preferentially express a desired product, as well as ADA" cells and ADA" cell lines. Use of cell lines that will process the protein more effectively or properly (e.g., by making-post translational modifications such as gammacarbox- ylation) is also possible. Brief Description of the Drawings

Figure 1 illustrates the structure of plasmid p9ADA5-29.

Figure 2 illustrates the structure of plasmid pFVXM.

Detailed Description of the Invention

According to the method of the present invention, a cell line containing an endogenous ADA gene is transformed with a foreign ADA cDNA. The production of ADA cDNA would follow a procedure analogous to that for cloning any other gene. [See generally Maniatis, T. et al.. Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory (1982); Toole, J. J. et al.. Nature 312; 342-47 (1984)]. The sequences of human ADA cDNA and mouse derived ADA cDNA have been determined [See Wiginton, D. A. et al., Nucl. Acids Res. 12; 1015-1024 (1984); Valerio, D. et al.. Gene 31: 147-153 (1984); Yeung, c. et al., J. Biol. Chem.. 258: 15179-15185 (1983) ]. ADA cDNA can be placed into a mammalian expression vector using techniques well known by those having ordinary skill in the art.

The cell to be transformed may be any ADA+ eucaryotic cell, including yeast protoplasts and various bacterial cells, but is preferably a nonfungal cell and most preferably, is a stable mammalian cell line. Useful in the practice of this invention are HeLa cells, melanoma cell lines such as the Bowes cell line, mouse L cells, mouse fibroblasts, mouse NIH 3T3 cells, and the like. Cell lines that are known to stably integrate ADA and other genes into their chromosomal DNA are also desirable, e.g., Chinese hamster ovary (CHO) cell lines, human hepatoma Hep G2 cell lines and mouse myeloma cell lines, depending upon the other requirements placed upon the cell line.

Exogenous genes are normally not expressed as well as endogenous chromosomal genes. It is thus a surprising aspect of the invention that it is possible to transform ADA+ cells with exogenous ADA and select for transformants charac¬ terized by significantly higher levels of ADA expression in comparison to endogenous ADA+ cells which undergo ADA gene amplification as a result of the same selection proced¬ ures. ADA is unique because in most cells it is expressed at a very low level. Introduction of an efficient expressing ADA gene renders those transformed cells capable of selec¬ tion. However, a few ADA+ cell lines express higher ADA levels than produced in most cell lines, e.g., those derived from gastrointestinal and thy us tissues, and should be avoided. [See Lee, P.A., Dev. Biol. 11: 227-233 (1973); Barton, R. et. al.. Cell Immunol. 49: 208-214 (1982); Sidi, Y. et. al., Thvmus 4: 147-154 (1982)]. The population of cells exposed to transforming conditions is then processed to identify the transformants, i.e., the small subpopulation which exhibit the phenotype of the ADA selection gene. The cells in the culture are screened for the phenotype by placing selection pressure on the cell. The specific selection method to be used can be determined by the person of ordinary skill in the art. Specific known methods for selecting for increased ADA expression are summarized below. The skilled artisan can adapt these and other known methods to select for cells containing exogenous ADA.

One such ADA selection method involves the use of adenosine analogues. Cells can be selected for resistance to cytotoxic adenosine analogues 9- -D-arabinofuranosyl adenine (Ara-A) or 9- -D-xylofuranosyl adenine (Xyl-A) . Multiple step selection in either Ara-A or Xyl-A results in cell populations with increased ADA activity. [See, Yeung, C. et. al., J. Biol.Chem. 258: 8330-8337 (1983)]. ADA has the ability to catalyze the irreversible conversion of these adenine analogues to their respective inosine deriva- tives which are eventually detoxified by removal of the ribose by purine nucleoside phosphorylase to yield hypo- xanthine. Because cells may become resistant to these analogues by loss of adenosine kinase activity, not all surviving cells will have increased levels of ADA. [V. L. Chan et. al.. Somatic Cell Genet. 7: 147-160 (1981); Yeung, et. al. supra]. However, the frequency of loss of adenosine kinase is usually low in cells which contain a diploid complement of the adenosine kinase gene.

A selection protocol which selects for the presence of adenosine kinase [Chan, T. et. al.. Somatic Cell Genetics 4.: 1-12 (1978)] has been modified so that it can also be used to select for increased expression of ADA. [See Yeung, C. et. al., supra 15179-15185 (1983)]. In contrast to the first procedure, all surviving cells exhibit increased levels of ADA. Adenosine kinase is selected for in the presence of AAU (adenosine, alanosine, uridine) . Under this growth condition, cells are blocked in de novo AMP (adenosine monophosphate) biosynthesis by alanosine and require adenosine kinase to convert adenosine to AMP. Since adenosine depletes phosphoribosylpyrophosphate (PRPP) which results in the inhibition of endogenous pyrimidine synthesis, the medium is supplemented with uridine. [See Green, H. et. al.. Science 182: 836-837 (1973); Ishii, K., et. al.. Cell Sci 12: 429-439 (1973)]. However, when the adenosine concentration is increased 11-fold (hereinafter

11-AAU selection) the high concentrations of adenosine become cytotoxic and ADA is required to alleviate the tox- icity. [See Fox, I.H. et. al., Ann Rev Biochem 47: 655-686

(1978)]. Once functional ADA is required for cell growth, (R)-deoxycoformycin (dCF), an antibiotic demonstrated to be a tight binding transition-state analogue inhibitor of ADA (kd=2.5 x 10~¹²), can be used to select for amplification of the ADA gene. [See Agarwal, R. P. et. al., Biochem. Pharmacol. 26: 359-367 (1977); Frieden, C. et. al., Biochem. 19: 5303-5309 (1980)]. For the cell to survive in these systems, ADA is required in higher levels than most cells produce. Growth of cells in 11-AAU in the presence of sequentially increasing concentrations of dCF, selects cells which contain a high degree of ADA expression as a result of amplification of the ADA gene. [See Yeung, C, suora at 8338-8345 (1983)].

Yet another selection method employs deoxyadenosine as a carbon source. Cells can also be made growth dependent on ADA activity by blocking purine de novo synthesis with azaserine and feeding cells 2-deox adenosine as a purine source. [See Fernandez-Mejia, et. al., J. Cell Physio. 120: 321-328 (1984)]. Deoxyadenosine is available as a general purine source only if converted to deoxyinosine by ADA. As a result, cells can be selected for increased ADA activityby growth in azaserinewith increasingconcentrations of dCF. The medium is supplemented with deoxycytidine. [See Thelander, L. et. al., Ann. Rev. Biochem. 48: 133-158 (1979) ]. A similar approach has been described by Hunt, S. W., et al., J. Biol. Chem. 258: 13185-13192 (1983), utilizing adenosine as the sole carbon source. Under these conditions, dCF resistant variants of Novikoff rate hepatoma cells which require functional ADA, were isolated by growing adenosine kinase-deficient cells in a medium containing adenosine as the sole carbon source with stepwise increasing concentrations of dCF. This procedure yields cells which have amplified the ADA gene 320-fold. [See also, Hoffee, P.A. et al.. Somatic Cell Genet. 8: 13185-1392 (1983)]. In any given population a certain number of cells containing an endogenous ADA gene will express a higher level of ADA than other cells. Thus, the degree of selection pressure will effect the sensitivity of distinguishing cells transformed with exogenous ADA from cells containing higher levels of ADA expression from an endogenous ADA gene. Accordingly, it is desired that one select for those cells expressing ADA at a five-fold increase and more preferably a ten-fold increase over that typically found expressed by cells containing endogenous ADA genes. Transformants exhibiting higher levels of ADA than endogenous ADA+ cells can be obtained by using vectors that result in more efficient expression of the heterologous gene. Cells can be transformed by use of a vector that contains both the ADA gene and the product gene as well as one or more other elements such as enhancers, promoters, introns, accessory DNA, a polyadenylation site and three prime non-coding regions. [See Clark, S.C. et al., Proc. Natl. Acad. Sci. USA 81: 2541-2547 (1984) ; see also Kaufman, R. J., Proc. Natl. Acad. Sci. USA 82: 689-693 (1985)]. These may be obtained from natural sources or synthesized by known procedures. Basically, if the components found in DNA are available in large quantity, e.g., components such as viral functions, or if they are to be synthesized, e.g., polyadenylation sites, large quantities of vectors may be obtained with appropriate use of restriction enzymes by simply culturing the source organism, digesting its DNA withan appropriate endonuclease, separatingthe DNAfragments and identifying the DNA containing the element of interest and recovering the same. Various vector systems including polyoma or retrovirus systems can be used provided they express the ADA produced by the exogenous ADA gene at a level above that expressed by cells containing endogenous ADA. Preferably 5-times greater expression is desired, more preferably 10-times. Two classes of vectors can be employed in trans¬ formation herein. Transformation with unlinked vectors, that is, one vector containing the exogenous ADA gene and another vector containing the desired exogenous product gene, can be accomplished simultaneously. Methods for facilitating cellular uptake of DNA are well known to those skilled in the art. Considerably better transformation efficiencies result from transformation with a molar excess of product gene to ADA gene, preferably on the order of 10:1 or higher. To most effectively obtain coamplification of ADA and product gene, the use of linked vectors in which the ADA and product genes are covalently bound is preferred. The coding strands of the ADA and product genes are preferably joined by directly ligating the product stop codon adjacent to the ADA gene start codon. The genes may be ligated through an oligodeoxyribonucleotide bridge. The bridge should be free of termination or start codons, and of palindromes to reduce the probability of forming RNA hairpin loops. Alternatively, one may transform with a vector or vectors containing a plurality of discrete product genes.

The vectors for use in producing the cells or cell lines useful in the method of the present invention are preferably supercoiled, double-stranded circular constructs, the form in which vectors are obtained from the standard prokaryotic cloning procedure. However, the vectors may be linearized, i.e., covalently cleaved at one point, incidental to other steps such as ligation to geno ic accessory DNA.

One preferred vector is plasmid p91023(B) which is deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD in E. coli MC1061 under ATCC deposit number 39754. The deposited vector can be modified by using EcoRl digestion to delete the CSF gene and replace it with an ADA gene. p91023(B) has been used for the expression of ADA in CHO cells and Baby Hamster Kidney cells, BHK.

As one embodiment of the invention, a vector containing a polyoma origin of replication and transcription enhancer in operative association with an exogenous ADA gene and exogenous gene coding for a desired protein, is provided. For example, the p90123 vector can be modified using tech- niques known in the art to delete the SV40 enhancer element and replace it with the polyoma, ADA and protein coding sequences. The resulting plasmid can respond to polyoma virus early T antigen and replicate in the presence of polyoma T antigen in mouse cells. This vector can then be introduced into a polyoma transformed mouse cell line which is expressing high levels of T antigen.

The polyoma system is analogous to that used in the COS system while having significant advantages thereover. COS cells are SV40 transformed monkey kidney cells, which express T antigen from SV40. Upon introduction of a plasmid that contains an origin of replication for SV40 into COS cells, the T antigen will act on that SV40 origin of repli¬ cation and will replicate very high copy numbers of the plasmid. Because the plasmid replicates to such a high copy number (about 50,000 copies per cell), the cells die rapidly and they can only be cultured for up to two weeks.

Polyoma replicates about an order of magnitude less efficiently than the COS system thereby providing better conditions for cell survival. Mouse cells in which polyoma can replicate, can be selected to express T antigen from polyoma. A plasmid which encodes for ADA and also has an origin of replication for the polyoma, can be introduced into the mouse polyoma transformed cells. Replication can occur as a plasmid rather than by integration and can range from 1,000 copies to 10,000 copies per cell. As a result of using a polyoma cell line and amplifying it using dCF in the presence of either high levels of adenosine or in the presence of Xyl-A, one should typically obtain a 100-fold higher resistance to dCF than is usually obtained in CHO or BHK.

In another embodiment of the present invention, a novel vector is provided which operatively links retrovirus sequences with an exogenous ADA gene. Group antigen, polymerase and envelope genes are deleted from the retrovirus and replaced with an ADA gene with the proper transcription and packaging signals to direct the envelopment of ADA gene into the virus. Such retrovirus construction techniques are known to those skilled in the art. This virus can then be transmitted from one cell to another cell. The presence of this ADA virus can be screened for by selecting for the presence of increased ADA expression in other cells. This vector is particularly desirable because it provides the capacity to get the ADA gene into cells with very high efficiency. The copy number may be amplifiable after the initial infection because of the presence of the ADA gene. Such retroviral vectors may be used to infect cells in vivo for use in mammalian gene therapy, as well as to create the cell lines useful in the present method. Once the host cell or cell line is transformed with a vector containing exogenous ADA DNA and an exogenous gene coding for a desired protein and desired transformants are selected, they are screened for ligation of the product gene into their chromosomes or for expression of the product itself. The product genes which can be used are essentially unlimited. Genes for proteins or enzymes having activities that are found in the cells of higher animals such as mammals ^"or vertebrae are the genes of most present interest herein. Even genes for proteins that may adversely affect the whole cell by synthesizing toxins or hydrolyzing host protein may be employed with procedural modifications, such as providing antitoxins in the culture medium or by selecting lower expression levels than would otherwise be optimum. Screening for ligation of the product gene can be accomplished using Southern blot analysis. Screening for expression of the product can utilize standard immunologi- cal, biological or enzymatic assays. Once the transformants have been identified, expression of the product gene can be amplified by subculturing in the presence of a selection agent in constant or increasing amounts as described above. Presently, the use of the 11-AAU procedure with increasing concentrations of dCF is preferred. Generally this entails (a) selecting one or more cells from the transformant cell population that express the product in a preferential fashion when compared to other cells in the population, (b) culturing the selected cell or cells to a subsequent cell population under conditions designed to select for a change in the expression of the phenotype, and (c) further selecting one or more cells from the subsequent cell population that express the product in a preferential fashion when compared to other cells in the subsequent population. Step (b) advantageously is conducted with a plurality of the step (a) clones. Although any of the procedures discussed supra can be utilized in both selection and amplification of the trans¬ formants, in more preferred embodiments, a combination of different procedures should be utilized. The Xyl-A procedure appears to be both more sensitive and more consistent than the 11-AAU system in selecting for uptake of exogenous DNA. Amplification of the transformants is preferably performed using the 11-AAU selection procedure.

Although the transformants can be grown in any medium, certain precautions are required depending upon the partic- ular procedure utilized as described below. For example, fetal calf serum has much higher levels of endogenous ADA than horse serum. In Xyl-A selection, 3nM dCF is used in the presence of 4.0uM Xyl-A in contrast to 11-AAU selection where O.OluM dCF is used with 0.03uM dCF in the presence of 1 mM adenosine. Thus when using a selection procedure that only requires very low levels of cytotoxic agent, e.g., Xyl-A, a growth media containing high levels of endogenous ADA, such as fetal calf serum, can detoxify the cytotoxic agent. If the use of fetal calf serum was desired, one could switch selection protocols to a different system, for example 11-AAU, which uses significantly more of a cytotoxic agent and would be minimally effected by fetal calf ADA. One could also utilize a separate selection marker.

Alternatively, if one desires to use the Xyl-A selection method, a number of strategies can be used to overcome the problem. Horse serum could be used instead of fetal calf serum because it does not contain high levels of endogenous ADA. However, if use of fetal calf serum is desired higher concentrations of Xyl-A can be utilized to minimize the effect of the fetal calf serum ADA. Further, one may add the Xyl-A right before selection and continue adding Xyl-A periodically to replace the Xyl-A detoxified by fetal calf ADA.

The following examples illustrate the use of the method of the present invention.

EXAMPLE 1 Construction of p9ADA5-29 and Expression of ADA cDNA in monkey kidney COS cells The ADA cDNA sequence for expression may be selected from the published human and murine sequences identified above. -For example, mouse ADA cDNA, pADA5-29 [See Yeung et al., supra at 15179-15185] was placed into a mammalian expression vector p90123, which is derived from p91023(B) by deleting the CSF gene with EcoRI digestion. The 1056 nuσleotide open reading frame in pADA5-29 was excised by Ncol and EcoRI digestion. The ends were filled in using Klenow fragment of DNA polymerase 1 and blunt-end ligated into the EcoRI site of vector p91023. The resultant vector, p9ADA5-29 (see Figure 1) , contains (from left to right) the adenovirus VA gene (VA) , the SV40 origin of replication including the 72 bp enhancer, the adenovirus virus major late promoter including the adenovirus tripartite leader and a 5' splice site (AdMLP) , a 3' splice acceptor site (3'ss), the ADA insert (ADA), the dihydrofolate reductase insert (DHFR) , the SV40 early polyadenylation site (SV40) and the pBR322 sequences needed for propagation in ___. coli. Vector p9ADA5-29, was used to transfect COS-1 cells using the DEAE-dextran procedure. [Kaufman, R. J., Proc. Natl. Acad. Sci. USA, supra] . Thetransfectedcellsunderwent zymogram analysis which indicated that the cells produced authentic mouse ADA at high levels.

EXAMPLE 2 Selection and Amplification of Cells TransformedwithADA cDNA

DHFR deficient CHO cells, CHO DHFR", (DUKXB11) , were grown in an alpha media with lOug/ml of thymidine, deoxy- adenosine and adenosine. Cells were transfected with pADA5-29 (25ug/10⁶ cells) as described by Kaufman, R. J., et al., J. Mol. Biol. 150:601-621 (1982). Forty-eight hours post-transfection, cells were plated (8xl0⁴ cells/lOcm plate) into either (1) alpha media supplemented with lOug/ml thymidine, 15ug/ml hypoxanthine, 4uM Xyl-A, with varying concentrations of dCF (2) alpha media supplemented with lOug/ml thymidine, lOug/ml deoxyadenosine, ImM uridine, l.OmM adenosine and varying concentrations of dCF. Four plates at each dCF concentration level were prepared for both media. The two media used correspond to the Xyl-A selectionprocedure and a modified 11-AAU selectionprocedure, 11-AU, respectively. The 11-AAU procedure was altered because CHO DHFR" cells cannot produce purines de novo. resulting in no need to use alanosine. To avoid detoxifi¬ cation of the cytological agents by the low levels of ADA endogenous to fetal calf serum, 10% fetal calf serum is added just prior to use of the media.

This transfection procedure was also repeated exactly as described above with no exogenous ADA DNA placed into the

CHO cell lines to produce mock-transfected CHO DHFR" cells for comparison. Results of the selection procedures showed that the Xyl-A selection media is more sensitive in indicating uptake of exogenous DNA than the 11-AU procedure. Selection for DNA uptake is preferably measured using about 4uM Xyl-A and about 0.003-0.OluM dCF.

Transformants were amplified using the 11-AAU procedure in combination with increasing levels of dCF as described in Yeung, C. et al., supra at 8338-8345, and as modified above by excluding alanosine. Transformants were maintained in DMEM supplemented with 10% fetal calf serum (Grand Island Biological Company) and incubated at 37°C. The transformed CHO DHFR" cells were grown in the 11-AU medium described above.

Six transformed colonies which were selected for by 11-AU selection at dCF concentrations of 0.03 and O.luM were placed in the above described media. These cells were then exposed to O.luM or 0.5uM of dCF respectively. Those cells not producing large amounts of ADA were killed. Once growth resumed for surviving cells, the cells were passaged several times at the same level of dCF. Then the dCF concentration was increased. Cells were exposed to dCF step-wise at levels of 0.03uM, O.luM, 0.5uM, luM, 5uM, and 20uM.

Cells to be analyzed were removed from drug selection for 1 week and fed with fresh DMEM plus 10% serum 24 hours before harvest. Cells were harvested by trypsinization, washed three items with Hank's balanced salt solution (without Mg²⁺ and Ca²⁺) , and resuspended in twice their packed volume of homogenizing medium (10 M Tris-HCl, pH 7.5, ImM beta -merσaptoethanol, and 1 mM EDTA) . The resus¬ pended pellet was frozen at -20°C, thawed and homogenized using a motorized Teflon homogenizer. The samples were centrifuged twice at 15,000 x g for 30 min to remove debris. The supernatants (containing ~lmg of protein/ml) were applied directly to starch gels. Electrophoresis was conducted at 4°C using 200V for 16 hours or 400V for 5 hours. Following electrophoresis, the starch gel was sliced into replica sheets of -1 mm thickness and histo- chemically stained for adenosine deaminase activity as described in Sicilano, M. J. , et al., Chromatographic and Electrophoretiσ Techniques (Smith, I., ed.) 4th Ed., vol 2, pp. 185-209 Wm. Heinemann Medical Books Ltd., London (1976); and Harris, H. et al.. Handbook of Enzyme Electrophoresis in Human Genetics. North/Howland, Oxford (1976) .

This treatment resulted in an amplification for the transformants selected at O.luM dCF of about 10-times and for the cells selected at 0.03uM dCF of about 50-times.

Further amplification is obtained by continuing to apply selection pressure on surviving cells with step-wise incre¬ ments of dCF as described above.

EXAMPLE 3

Transformation and Coamplification of ADA with a Product Gene

Plasmid p9ADA5-29, described in Example 1, is mixed with a p91023 (B) derivative, p91023-p, containing a DNA sequence coding for the desired product polypeptide instead of the CSF gene. 50 ug p91023-p is mixed with 0.5 ug P9ADA5-29 and precipitated by the addition of NaOAσ (pH 4.5) to 0.3 M and 2.5 vols. of ethanol. Precipitated DNA is allowed to air dry, then resuspended in 2X HEBSS (.5ml) [Chu et al.. Gene .13: 197-202 (1981)] and mixed vigorously with .25 M CaCl2 (.5ml) as described in Kaufman, R. J. et al. , J. Mol. Biol. supra. The calcium-phosphate-DNA pre¬ cipitate is allowed to sit 30 minutes at room temperature, and applied to CHO DUKX-B1 cells [Chasin, et al., Proc. Natl. Acad. Sci. USA 77: 4216-4220 (1981) ] . The growth and maintenance of these cells has been described in Kaufman et al., J. Mol. Biol. supra and Chasin et al., supra.

The DUKX-B1 cells are subcultured at 5 x 10⁵/10^cm dish for 24 hours prior to transfection. The media is removed, and the DNA - calcium phosphate precipitate is added to the monolayer. After 30 minutes incubation at room temperature. 5ml of alpha-media (Flow) with 10% fetal calf serum is applied and the cells are incubated at 37°C for 4.5 hours. The media is then removed from the monolayer of cells, 2ml of alpha-media (Flow) containing 10% glycerol is added for 3 minutes at room temperature (24°C) and then removed and the cells are rinsed and fed with alpha-media containing 10% fetal calf serum, 10 ug/ml each of thymidine, adenosine, deoxyadenosine, penicillin and streptomycin. Two days later the cells are subcultured 1:15 in the selection media as described above.

Colonies will appear 10-12 days after subculturing into selective media. Two schemes for selection and amplifi¬ cation can be followed. In the first scheme single indepen¬ dent cloned transformants are isolated on the basis of uptake of the exogenous ADA DNA and subsequently each clone is propagated under conditions to increase expression of the product gene i.e., growth in increasing concentrations of dCF. In the second scheme pools of multiple independent transformants are isolated on the basis of uptake of the exogenous ADA DNA and are propagated under conditions to increase expression of the product gene, i.e., growth in increasing concentrations of dCF. Then individual clones are isolated from the mass selected population and analyzed for expression of the product gene. Those clones exhibiting highest levels of product gene expression are grown again under conditions to further increase product expression (i.e., growth in increasing concentrations of dCF in the culture media) .

An alternative method of transfecting and coamplifying ADA or a product gene is to employ only a p91023 vector containing both the ADA gene and the product gene in place of the unlinked vectors p91023-p and p9ADA5-9 in the pro¬ cedures of this example. EXAMPLE 4 Selection for Heterologous ADA Genes inMouse Fibroblast cells

A plasmid, pXC-ADA, containing the polyoma virus origin of replication and transcriptional enhancer in place of the SV40 origin and transcriptional enhancer in pADA5-29 was derived by the following procedures. Starting plasmid p.84.A2.X containing the polyoma regulatory region ligated with an Xhol linker at the Bel 1 site [See Veldman et al., Mol. Cell Biol. 5.:649-658 (1985)] was digested with the restriction endonuclease Bgl 1. The end was rendered flush by a fill-in reaction using T4 DNA polymerase 1 in the presence of 100 uM each dATP, dTTP, dCTP, and dGTP [Maniatis et al. supra] . EcoRI linkers (Collaborative Res.) were applied and the DNA digested with an excess of EcoRI and Xhol. The resultant DNA was electrophoresed on a 6% poly- acrylamide gel using Tris-Borate as a buffer system and the fragment migrating at 370 bases was isolated by electro- elution (Id.) . The 370 bp fragment was ligated to vector pAdD26SVpA#l, described in Kaufman, R. J. et al. Mol. Cel. Biol.. supra which was previously digested with Xhol and EcoRI. The resultant plasmid was used to liberate an approximately 400 bp fragment by Xhol and Cla 1 digestion. This fragment, containing 24 bp from pBR322 between the EcoRI site to the Cla 1 site, was isolated and ligated to pADA5-29 which had been previously digested with Xhol and Cla 1. The DNA was used to transform E. coli HB 101 for tetracycline resistance and colonies were screened by filter hybridization [Grunstein et al. Proc. Natl. Acad. Sci.. 72: 3961 (1975)] to a probe prepared by nick translation of the original Xhol-Bgl 1 fragment from p.84.A2.X. Positively hybridizing clones were analyzed and plasmid pXC-Ada was prepared by banding DNA twice in cesium chloride. The structure of plasmid pXC-Ada was confirmed by analysis after digestion with multiple restriction enzymes. pXC-Ada was transfected into mouse fibroblasts previ¬ ously transformed with an origin defective polyoma virus early region (MOP, provided by Claudio Basilico, N.Y. Uni¬ versity School of Medicine) as described by Kaufman, et al. J. Mol. Biol. , supra except the cells were propagated in DME media with 10% fetal calf serum.

The early region of polyomavirus expresses three trans- formation antigens (large, middle, and small T antigens) which elicit the transformed phenotype. Large T antigen elicits replication of .plasmids introduced into the mouse fibroblasts containing a polyoma origin of replication. [Tyndall et al., Nuσ. Acids Res.. £:6231-6250 (1981)]. Forty-eight hours after transfection, cells were subcultured at 2X10⁵ cells/dish in media containing 4uM Xyl-A with increasing concentrations of dCF. Five plates at each concentration were prepared.

After two weeks, both cells transfected with pXC-ADA and mock transfected (no exogenous DNA) had colonies upon selection in O.OluM dCF. In 0.03uMdCF, 43 colonies appeared in the transfected compared to 3 in the mock. This number decreased for transfected cells to 34 at O.luM dCF and to 15 at 0.3uM dCF. In 0.3uM dCF, 43 colonies appeared in the transfected compared to 3 in the mock. This number decreased for transfected cells to 34 at O.luM dCF and to 15 at 0.3uM dCF. Virtually no colonies were found at these higher levels in the mock cells. Growth of cells at these high concentrations of dCF indicates that the transfected cells have many copies of the plasmid pXC-ADA even without amplifi¬ cation by sequential selection in higher concentrations of dCF. Use of pXC-ADA to select for high levels of ADA expression in polyoma transformed in fibroblasts most likely resulted from high plasmid replication driven by the polyoma replication signals.

EXAMPLE 5 Selection for Expression of Retrovirus

Transmitting Functional ADA The retroviral vector pEVX [Kriegler et al.. Cell, 38: 483-491 (1984)] was derived from sequences of both Moloney leukemia virus and Harvey Sarcoma virus. pEVX was modified by deletion of the Harvey Sarcoma virus packaging site while still retaining the packaging signal sequences of Moloney leukemia virus which are fully functional r roc. Natl. Acad. Sci. 72:3961 (1975)].

The resulting plasmid pFVXM [Fig. 2] contains the viral long terminal repeats (LTRs) , and an internal polylinker for insertion of heterologous genes. It does not contain the retroviral group antigen (gag) , polymerase (pol) , and envelope (env) genes. The Bgl II site in this plasmid is unique and is ideal for the insertion and subsequent expres- sion of virions capable of producing the protein encoded by the inserted squence.

Exogenous ADA was prepared for insert into pFVXM, by digesting pADA5-29 with EcoRI and Sacl, treating with T4

DNA polymerase to flush the ends, and applying Bgl II linkers (Collaborative Res.). After Bgl II digestion and agarose gel electrophoresis, on approximately 1.8 kb band was isolated. This fragment was ligated to pFVXM, which had previously been digested with Bgl II. Colonies were screened by colony hybridization (Grunstein et al. supra.) to a nick-translated DNA fragment (the original EcoRI and

Sacl fragment isolated from pADA5-29) . DNA was prepared frompositivelyhybridizingclones byrestrictionendonuclease analysis. One clone, pRetro ADA-1-1, was found to contain the ADA insert in the proper orientation with respect to the retroviral long terminal repeat (LTR) used for tran¬ scription initiation. pRetro ADA 1-1 DNA was prepared by propagation in E. coli HB101 and DNA banded twice in cesium chloride. The DNA was transfected into mouse fibroblast ψ 2 cells [Mann et al.. Cell 33: 153-159 (1983)] which contain a defective Moloney viral genome that cannot be packaged into mature virions. However, the gag, pol, and env polypeptides (which are required for virus production and are missing from pRetro ADA 1-1) are expressed from the defective genome. Those proteins are sufficient to complement all functions missing in pRetro ADA 1-1. 48 hours after CaPθ4 mediated DNA transfecton of 2xl0⁶ ψ2 cells with 25g of pRetro ADA 1-1, the cells were subcultured into 4uM Xyl-A with 0.01M dCF. Three colonies appeared from the cells receiving DNA where no colonies appeared when the DNA was omitted. One colony, ψ 2-ADA, was chosen and analyzed for ADA retrovirus production.

The conditioned media from 10⁶ cells (1 ml) was harvested after 24 hours and after filtration (0.2uM filter) applied to 3T3 cells (2xl0⁶) in the presence of 8ug/ml of polybrene for 2 hours. The virus was then removed and the cells were supplied with fresh media. 48 hours later the confluent 3T3 cells were subcultured 1:10 into media con- taining 4uM Xyl-A and 0.01 or 0.03uM dCF. After 14 days colonies were counted. The uninfected cells had no colonies growing in 0.01 or 0.03uM dCF per 2xl0⁶ originally infected cells. Infected cells had approximately 4000 colonies in O.OluM dCF and 3000 colonies in 0.03uM dCF. These results indicate that >10³ infectious units were present per ml of culture fluid from the transfectedψ 2 cells.

This procedure allows the introduction of an amplifiable vector into cells with a potent selection system to obtain cells expressing the heterologous ADA. It should be possible by using techniques well known in the field to introduce other genes into the retrovirus in order to also place them into cells. The presence of the exogenous ADA gene allows for potential amplification of the inserted viral DNA. In addition the amplification of the retroviral sequences in the ψ2 cells allows for production of higher titre virus stocks which are essential in order to introduce genes into animals and into humans.

Claims

What is claimed is:

1. A method for producing high level expression of a selected exogenous protein comprising culturing a cell which comprises at least one copy of an endogenous gene coding for ADA, amplified copies of an exogenous gene coding for ADA and amplified copies of an exogenous gene coding for said selected protein.

2. The method according to claim 1 further comprising transforming a cell containing an endogenous gene coding for ADA with an exogenous gene coding for ADA and an exogenous gene coding for said selected protein and coamplifying said exogenous ADA gene with said exogenous protein gene.

3. Themethod accordingto claim 2, furthercomprising transforming said cell with a single expression vector comprising said exogenous protein gene and exogenous ADAgene.

. Themethod according to claim 3, furthercomprising transforming said cell with a single expression vector on which said exogenous protein gene and said exogenous ADA gene are covalently linked.

5. The method according to claim 2, furthercomprising transforming said cell with one expression vector comprising said exogenous ADA gene and second expression vector com¬ prising said exogenous protein gene.

6. The method according to claim 1, wherein said cell is selected from the group consisting of yeast cell, bacterial cell and mammalian cell lines.

7. The method according to claim 6 wherein said mammalian cell lines are selected from the group consisting of Bowes cell line, mouse L cells, mouse fibroblasts, mouse NIH 3T3 cells, human hepatoma Hep G2 cell lines and CHO cell lines.

8. A cell line for use in producing high levels of expression of a selected exogenous protein produced by transforming a cell line which contains an endogenous gene coding for ADA with an exogenous gene coding for ADA and an exogenous gene coding for said protein and co¬ amplifying said exogenous ADA and protein genes.

9. The cell line according to claim 8, wherein said exogenous gene coding for ADA is selected from the group consisting of murine ADA, human ADA, bacterial ADA and yeast ADA.

10. A vector comprising an exogenous gene coding for ADA in operative association with retrovirus transcription and packaging sequences capable of directing the envelopment of said gene.

11. The vector according to claim 10, further com¬ prising a gene encoding a desired exogenous gene.

12. A vector comprising an exogenous gene coding for

ADA and a gene coding for a desired protein in operative association with an adenovirus VA gene, an SV40 origin of replication, an adenovirus major late promoter and an SV40 early polyadenylation site.

13. A vector comprising an exogenous gene coding for ADA, and a gene coding for a desired protein in operative association with a polyoma virus origin of replication and polyoma virus transcriptional enhancer.