| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 6, 1999 |
| PATENT TITLE |
Recombinant DNA methods vectors and host cells |
| PATENT ABSTRACT |
The present invention relates to vectors useful for transforming a lymphoid cell line to glutamine independence. The vectors comprise an active glutamine synthetase (GS) gene as well as a heterologous gene of interest to be expressed. The preferred embodiments encompass vectors wherein the heterologous gene is expressed from a relatively strong promoter and the GS gene is expressed from a relatively weak promoter. In one example, the heterologous gene is operatively linked to the hCMV-MIE promoter and the GS gene is operatively linked to the SV40 early region promoter. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | January 23, 1995 |
| PATENT REFERENCES CITED |
Foeching et al, Gene 45: 101, 1986. Alberts et al., "Molecular Biology of the Cell," Garland Publishing, Inc., New York, pp. 184-193 (1983). Watson et al., "Recombinant DNA, A Short Course," Freeman & Co., pp. 50-90 (1983). Sanders, Peter G. et al., "Amplification and cloning of the Chinese hamster glutamine synthetase gene," The EMBO Journal, vol. 3, No. 1, pp. 65-71 (1984). Pennica, Diane et al., "Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli", Nature, vol. 301, pp. 214-221, Jan. 20, 1993. Donn, Gunter et al., "Herbicide-Resistant Alfalfa Cells An Example of Gene Amplicicaton in Plants," Journal of Molecular and Applied Genetics, pp. 621-635 (1984). Young, Anthony P. et al., "Mouse 3T6 Cells That Over-Produce Glutamine Synthetase," The Journal of Biological Chemistry, vol. 258, No. 18, pp. 11260-11266 (Sep. 25, 1983). de Saint Vincent, Bruno Robert et al., "The Cloning and Reintroduction into Animal Cells of a Functional CAD Gene, a Dominant Amplifiable Genetic Marker," Cell, vol. 27, pp. 267-277 (Dec. 1981). Murray, Mark J. et al., "Construction and Use of a Dominant, Selectable Marker: a Harvey Sarcoma Virus-Dihydrofolate Reductase Chimera," Molecular and Cellular Biology, vol. 3 , No. 1 pp. 32-43, Jan. 1983. Kaufman, Randal J. et al., "Selection and amplification of heterologous genes encoding adenosine dreaminase in mammalian cells," Proc. Natl. Acad. Sci. USA, vol. 83, pp. 3136-3140 (May 1986). Kaback, David B. et al., "Ribosomal DNA Magnification in Saccharomyces cerevisiae," Journal of Bacteriology, vol. 134, No. 1, pp. 237-245 (Apr. 1978). Vel 'kov, V. V., "Amplification of Genes in Prokaryotic and Eukaryotic Systems," Soviet Genetics, vol. 18, pp. 384-396 (1982). Sambrook, J. et al. Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, pp. 16.45 to 16.46 (1989). Foecking, Mary K. et al., "Powerful and versatile enhancer-promoter unit for mamalian expression vectors," Gene 45, pp. 101-105 (1986). Review of Physiological Chemistry, 16th Edition, Chapter 23--p. 369, Lange Medical Publications, (1977). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of conferring glutamine independence to a myeloma cell line, comprising transforming said myeloma cell line with a vector comprising a GS gene and a gene or genes encoding a protein(s) heterologous to said myeloma cell line, wherein the genes are arranged such that said GS gene can be expressed and glutamine independent myeloma colonies can be produced. 2. The method of claim 1, wherein said gene or genes encoding the protein(s) heterologous to said myeloma cell line comprises a relatively strong promoter, and wherein said GS gene comprises a relatively weak promoter located upstream of said gene or genes encoding the protein(s) heterologous to said myeloma cell line so that transcription of the heterologous gene or genes does not run through the GS gene. 3. The method of claim 2, wherein said relatively weak promoter is the SV40 early region promoter and said relatively strong promoter is the hCMV-MIE promoter. 4. The method of claim 1, wherein said gene or genes encoding the protein(s) heterologous to said myeloma cell line comprises a relatively strong promoter, and wherein said GS gene comprises a relatively weak promoter that directs expression in the opposite direction to that of said gene or genes encoding the protein(s) heterologous to said myeloma cell line. 5. The method of claim 4, wherein said relatively weak promoter is the SV40 early region promoter and said relatively strong promoter is the hcMV-MIE promoter. 6. The method of claim 4, wherein said vector comprises a GS gene that comprises a weak promoter, and wherein said gene or genes encoding the protein(s) heterologous to said myeloma cell line comprises an Ig heavy chain gene having a strong promoter and an Ig light chain gene having a strong promoter, wherein said strong promoter of said light chain gene is oriented in the opposite direction to said promoters of said GS and heavy chain genes, and wherein said Ig heavy chain gene is downstream from said GS gene. 7. The method of claim 1, wherein said vector comprises a GS gene that comprises a weak promoter, and wherein said gene or genes encoding the protein(s) heterologous to said myeloma cell line comprises an Ig heavy chain gene having a strong promoter and an Ig light chain gene having a strong promoter, wherein said strong promoter of said Ig light chain gene is orientated in the opposite direction to said promoters of said GS and heavy chain genes, and wherein said Ig heavy chain gene is downstream from said GS gene so that transcription of the heterologous gene does not run through the GS gene. 8. The method of claim 1, wherein said GS gene comprises a weak promoter, wherein said gene or genes encoding the protein(s) heterologous to said lymphoid cell line comprises an Ig light chain gene having a strong promoter and an Ig heavy chain gene having a strong promoter, wherein said GS gene, Ig light chain gene, and Ig heavy chain gene are transcribed in the same direction, and wherein said GS gene is located upstream of said Ig light chain gene and said Ig heavy chain gene so that transcription of the heterologous gene(s) does not run through the GS gene. 9. The method of claim 1 wherein the GS gene is expressed from an SV40 early region promoter. 10. The method of claim 1 wherein the GS gene is expressed from an hCMV-MIE promoter. 11. The method of claim 9 wherein the GS gene and promoter are derived from pSV2GS. 12. The method of claim 10 wherein the GS gene and promoter are derived from pCMGS. 13. The method of claim 1 wherein all genes are expressed from the same type of promoter. 14. The method of claim 13 wherein the type of promoter is the hCMV-MIE promoter. 15. The vector of claim 14 wherein the vector is derived from pCMGS. 16. The vector of claim 8 wherein the vector is pSV2GScLccHc or pST6. 17. A method of selecting myeloma cells transfected with a vector comprising a GS gene and a gene or genes encoding a protein(s) heterologous to said myeloma cells, comprising: (i) plating transfected cells in one volume of non-selective medium containing glutamine; (ii) after 24 hours, adding two volumes of glutamine-free medium; and (iii) recovering myeloma colonies after 7 days incubation. 18. A method of conferring glutamine independence to a lymphoid cell line, comprising transforming said lymphoid cell line with a vector comprising a GS gene and a gene or genes encoding a protein(s) heterologous to said lymphoid cell line, wherein the genes are arranged such that said GS gene can be expressed and glutamine independent lymphoid colonies can be produced. 19. The method of claim 18, wherein said gene or genes encoding the protein(s) heterologous to said lymphoid cell line comprises a relatively strong promoter, and wherein said GS gene comprises a relatively weak promoter located upstream of said gene or genes encoding the protein(s) heterologous to said lymphoid cell line so that transcription of the heterologous gene or genes does not run through the GS gene. 20. The method of claim 19 wherein said relatively weak promoter is the SV40 early region promoter and said relatively strong promoter is the hCMV-MIE promoter. 21. The method of claim 18, wherein said gene or genes encoding the protein(s) heterologous to said lymphoid cell line comprises a relatively strong promoter, and wherein said GS gene comprises a relatively weak promoter that directs expression in the opposite direction to that of said gene or genes encoding the protein(s) heterologous to said lymphoid cell line. 22. The method of claim 21, wherein said relatively weak promoter is the SV40 early region promoter and said relatively strong promoter is the hCMV-MIE promoter. 23. The method of claim 21, wherein said vector comprises a GS gene that comprises a weak promoter, and wherein said gene or genes encoding the protein(s) heterologous to said lymphoid cell line comprises an Ig heavy chain gene having a strong promoter and an Ig light chain gene having a strong promoter, wherein said strong promoter of said light chain gene is oriented in the opposite direction to said promoters of said GS and heavy chain genes, and wherein said Ig heavy chain gene is downstream from said GS gene. 24. The method of claim 18, wherein said vector comprises a GS gene that comprises a weak promoter, and wherein said gene or genes encoding the protein(s) heterologous to said lymphoid cell line comprises an Ig heavy chain gene having a strong promoter and an Ig light chain gene having a strong promoter, wherein said strong promoter of said Ig light chain gene is orientated in the opposite direction to said promoters of said GS and heavy chain genes, and wherein said Ig heavy chain gene is downstream from said GS gene so that transcription of the heterologous gene does not run through the GS gene. 25. The method of claim 18, wherein said GS gene comprises a weak promoter, wherein said gene or genes encoding the protein(s) heterologous to said lymphoid cell line comprises an Ig light chain gene having a strong promoter and an Ig heavy chain gene having a strong promoter, wherein said GS gene, Ig light chain gene, and Ig heavy chain gene are transcribed in the same direction, and wherein said GS gene is located upstream of said Ig light chain gene and said Ig heavy chain gene so that transcription of the heterologous gene(s) does not run through the GS gene. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to methods for improving the usefulness of lymphoid cell lines as host cells for the production of proteins by recombinant DNA technology. The present invention also relates to vectors for use in such methods and to host cells produced by such methods. 2. Description of the Prior Art Lymphoid cell lines are at present being appraised for use as host cells in the production by recombinant DNA technology of immunoglobulin molecules, related hybrid or chimeric proteins (Ig-type molecules), or other recombinant proteins. Since the lymphoid cells include myeloma cells which are of the same general type as the B cells which produce Ig molecules In vivo, it is envisaged that they will naturally possess the intracellular mechanisms necessary to allow proper assembly and secretion of Ig-type molecules. Such lymphoid cell lines may also be of use in the production by recombinant DNA technology of non-Ig-type molecules. It is known that many lymphoid cell lines, such as myeloma cell lines and T cell lymphomas, cannot be grown in vitro on media lacking in glutamine. It has been suggested that it would be useful to be able to transform lymphoid cell lines to glutamine independence, since this may provide an advantageous method for selecting transformed cell lines. It has been conjectured that such a cell line could be transformed to glutamine independence by incorporating therein a gene coding for glutamine synthetase (GS). Such a suggestion is made in EP-A-0 256 055 (Celltech). However, it has subsequently been found that hybridoma cell lines can generate spontaneous variants able to grow in a glutamine-free medium at such a high frequency that the identification of transfectants is difficult or impossible. For myeloma cell lines, transfection with a GS gene and growth of the transformed cells in a glutamine-free medium does not result in significant survival rates. SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method for transforming lymphoid cell lines to glutamine independence. According to the present invention, there is provided a method for transforming a lymphoid cell line to glutamine independence which comprises: transforming the lymphoid cell line with a vector containing an active glutamine synthetase (GS) gene; growing the transformed cell line on a medium containing glutamine; and continuing the growth of the transformed cell line on a medium in which the glutamine is progressively depleted or on a medium lacking glutamine. Preferably, the lymphoid cell line is a myeloma cell line. Preferably, the glutamine-depleted or glutamine-free medium contains asparagine. Alternatively the medium contains another nutrient which enables the transformed cell line to survive on a glutamine free medium. This other nutrient may be an ammonia donor, such as ammonium chloride. It has surprisingly been found that if the transformed lymphoid cell line is not firstly grown on a glutamine-containing medium, it is not possible to obtain the growth of any cell line, whether or not it has been transformed by the vector. By use of the method of the present invention, it is possible to select for lymphoid cell lines which have been transformed by the vector. Alternatively, the lymphoid cell line may be transformed with a vector containing both an active GS gene and a gene encoding another selectable marker, such as a gpt gene, or cotransformed with separate vectors encoding GS and the selectable marker respectively. Transformed host cells can then be selected using the selectable marker prior to depletion of glutamine in the medium. The advantage of this method is that it enables selection for vector maintenance to be achieved without the use of a toxic drug. Host cells in which the vector is eliminated will not be able to survive in a glutamine-free medium. A further advantage of this method is that it enables selection for gene amplification to be carried out without the risk of amplification of the host cell's endogeneous GS genes. Preferably the glutamine in the medium is progressively depleted by dilution with a medium containing aspargine but lacking glutamine. Preferably, the vector used to transform the lymphoid cell line also contains an active gene coding for a protein heterologous to the lymphoid cell line. Alternatively, the lymphoid cell line may be co-transformed with a separate vector containing the active gene coding for the heterologous protein. The heterologous protein may be one which is expressed as a single chain (although it may be cleaved after expression into a multichain protein). Examples of such single chain expression products are tissue plasminogen activator (tPA), human growth hormone (hGH) or tissue inhibitor of metalloproteinase (TIMP). Preferably, however, the heterologous protein is an Ig-type molecule. Such molecules require the separate expression of two peptide chains which are subsequently assembled to form the complete molecule. Thus, the cell line will need to be transformed with active genes which encode separately a heavy chain (or heavy chain analog) and a light chain (or light chain analog). Preferably, the genes encoding the heavy and light chains are both present on the same vector as the GS gene. Alternatively, the vector containing the GS gene may have one of the heavy or light chain genes thereon, the other gene being on a separate vector. In a second alternative, the light and heavy chain genes are not present on the vector containing the GS gene but are present on the same or different vectors. The expression of such heterologous proteins may be substantially increased by subsequent selection for GS gene amplification, for instance using methionine sulphoximine (MSX) as the selection agent. It is preferred that the GS gene comprises a relatively weak promoter and that the gene (or genes) encoding the heterologous protein comprises a relatively strong promoter so that in the transformed cell lines, protein synthesis is directed preferentially to the production of the heterologous protein or peptide rather than to the production of GS. Moreover, a lower concentration of selection agent, such as MSX, will be required to select for gene amplification if the GS gene is controlled by a weak, rather than a strong, promoter. It is also conjectured that use of a weak promoter may enable the selection of transformed cell lines wherein the GS gene has been inserted at a particularly advantageous location in the genome. This will ensure that both the GS gene and any heterologous genes will be transcribed efficiently. It has been found that, in the preferred case, where all the genes are present on the same vector, it is necessary to design the vector carefully in order to achieve proper expression of the genes. Thus, according to a second aspect of the present invention, there is provided a vector for transforming a lymphoid cell line to glutamine independence and to enable it to produce a heterologous protein, the vector comprising a GS gene and a gene encoding the heterologous protein, wherein the vector is arranged such that expression of the GS gene is not hindered by transcriptional interference from the promoter/enhancer transcribing the sequence coding for the heterologous protein to such an extent that glutamine-independent colonies cannot be produced. Preferably, the genes on the vector are arranged in such orientations and with such promoters as substantially to prevent transcriptional interference. For instance, the GS gene may contain a relatively weak promoter, the gene encoding the heterologous protein may contain a relatively strong promoter, and the promoter of the GS gene may be located upstream of or may direct expression in the opposite direction to that of the gene encoding the heterologous protein. It has surprisingly been found that if the vector arrangement set out above is adopted, the GS gene is expressed in sufficient quantity to enable selection to be made and the heterologous protein is expressed more efficiently than with other vector arrangements. It has been observed that other vector arrangements, for instance using different promoters or a different ordering or orientation of the genes, can lead to a much reduced or even non-existent level of GS or heterologous protein production. It is conjectured (although the applicants do not wish to be limited to this theory) that if a gene containing a strong promoter is located upstream of a GS gene having a weaker promoter, the transcription of the upstream gene will run through into the downstream gene, thus producing occlusion of the downstream promoter. Since the frequency of transformed colonies is critically dependent on the level of GS gene expression, such promoter occlusion dramatically reduces the frequency with which transfectants are recovered. A preferred combination for the weak and strong promoters is the SV40 early region and the hCMV-MIE promoters. (hCMV-MIE=human cytomegalovirus major immediate early gene). However, other suitable promoter combinations will be readily apparent to those skilled in the art. A particularly preferred embodiment of the vector of the present invention comprises a GS gene having a weak promoter having downstream therefrom a heavy chain-like gene having a strong promoter, there being on the vector a light chain-like gene having a strong promoter oriented in the opposite direction to the promoters of the GS and heavy chain-like genes. Alternatively, promoter occlusion may be prevented by use of transcription terminator signals between the genes. In another alternative, the genes may be arranged with a unique restriction site between them. This site can then be used to linearise the vector before it is incorporated into the host cell. This will ensure that in the transformed host cell no promoter occlusion can take place. It will be appreciated that if the vector contains more than one gene encoding a heterologous protein, it will be necessary to ensure that none of the genes in the vector can promote transcriptional interference. For instance, if the vector contains a GS gene, a heavy chain gene and a light chain gene, it is preferred that either all three genes are transcribed in the same direction and that the GS gene is upstream of the other two genes or that the GS gene and one of the other genes are transcribed in the same direction, the GS gene is upstream of the first other gene, and the second other gene is transcribed in the other direction, and the promoter of the second other gene is located adjacent the promoter of the GS gene. The vector may comprise a viral vector, such as lambda phage, or a plasmid vector, for instance based on the well known pBR322 plasmid. However, any other of the vectors well known in the art may be adapted by use of conventional recombinant DNA technology for use in the present invention. The present invention also includes host cells produced by the method of the invention or containing vectors according to the invention. In particular, the present invention includes a lymphoid cell line which has been cotransformed with a vector containing a GS gene and a vector containing a gene encoding a heterologous protein, the vectors being arranged to ensure that the GS gene is not hindered by transcriptional interference to such an extent that glutamine-independent colonies cannot be produced. The present invention is described below by way of example only with reference to the accompanying drawings in which: BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an analysis of proteins secreted by NSO cells transfected with plasmid pAB2GS by Western blotting in which 25 .mu.l of culture supernatant or control tissue culture medium was run on a 10% SDS reducing polyacrylamide gel, blotted onto nitro-cellulose and probed with antisera recognising human Ig chains and then with .sup.125 I-labelled protein A; FIG. 2 shows the structure of plasmid pSV2GScLc; FIG. 3 shows the structure of plasmid pST6; and FIG. 4 shows a Southern blot analysis of genomic DNA from cell lines SV2GSNSO and CMGSNSO. In FIG. 1 of the drawings lane 1 shows purified chimeric B72.3 antibody to show the position of Ig light and heavy chains, lanes 2 to 5 show culture supernatants from four different transfected clones, and lane 6 shows culture medium as a negative control. In FIG. 2, E is the SV40 early region promoter, GS is a GS cDNA coding sequence, intron+PA is the small t-intron and the early region polyadenylation signal of SV40, BCMV is the hCMV-MIE promoter-enhancer, CLC is the coding sequence for the chimeric L-chain of a humanised antibody known as B72.3, and pA is the SV40 early polyadenylation signal. In FIG. 3, hCMV is the hCMV-MIE promoter enhancer (2.1 kb) fragment. CHC is the chimeric heavy chain coding sequence of the B72.3 antibody. CLC is the chimeric light chain coding sequence of the B72.3 antibody. Poly A contains the SV40 early polyadenylation signal. I+PA contains the small t intron of SV40 and the early region polyadenylation signal. SVE is the SV40 early promoter. A bacterial plasmid origin of replication and ampicillin resistance gene are provided by pBR322. FIG. 4 shows a copy number analysis of GS-vectors in NSO cells before and after selection with MSX. DNA samples were digested with BglI and BglII, electrophoresed on a it agarose gel, transferred to nitrocellulose and probed with the 0.5 kb 5' Pst1 DNA fragment of pGSC45 ›7! isolated from a GS cDNA. DNA samples are as follows: ______________________________________ Lane 1 plasmid pSV2GS equivalent to 100 copies/cell Lane 2 plasmid pSV2GS equivalent to 10 copies/cell Lane 3 plasmid pSV2GS equivalent to 1 copy/cell Lane 4 10 .mu.g NSO genomic DNA Lane 5 10 .mu.g SV2GSNSO genomic DNA Lane 6 10 .mu.g SV2GSNSO (100 .mu.M MSX resistant) genomic DNA Lane 7 plasmid pCMGS equivalent to 100 copies/cell Lane 8 plasmid pCMGS equivalent to 10 copies/cell Lane 9 plasmid pCMGS equivalent to 1 copy/cell Lane 10 10 .mu.g CMGSNSO genomic DNA Lane 11 10 .mu.g CMGSNSO (100 .mu.M MSX resistant) genomic DNA m.w. .lambda. phage DNA digested with Clal; molecular weight markers ______________________________________ A list of references is given at the end of the description. In the following, the references are indicated by numbers enclosed in square brackets. VECTORS In the following Examples, for comparative purposes, two plasmids described in EP-A-0 256 055 were used. These are plasmids pSVLGS1 and pSV2GS. Plasmid pSVLGS1 contains a GS minigene, containing cDNA and genomic DNA sequences, under the control of a SV40 late region promoter. Plasmid pSV2GS contains a cDNA sequence encoding GS under the control of a SV40 early region promoter. A vector pSV2BamGS was produced by converting the unique PvuII site in pSV2GS to a BamHI site by the addition of a synthetic oligonucleotide linker. By use of synthetic oligonucleotide linkers, the major immediate early gene promoter, enhancer and complete 5'-untranslated sequence from human cytomegalovirus (hCMV-MIE) (the Pst-1m fragment ›1! together with a synthetic oligonucleotide to recreate the remaining 5' untranslated sequence) was inserted between the NcoI sites of pSV2GS such that the hCMV-MIE promoter directs expression of the GS coding sequence. The resulting plasmid was labelled pCMGS. Plasmid pSV2BamGS was digested with BamHI to give a 2.1 kb fragment containing the transcription cassette. For convenient construction of other expression plasmids, a basic vector pEE6 was used. Plasmid pEE6 contains the XmnI to BclI fragment of plasmid pCT54 ›2! with the polylinker of plasmid pSP64 ›3! inserted between its HindIII and EcoRI sites but with the BamHI and SALI sites removed from the polylinker. The BclI to Bam HI fragment is a 237bp SV40 early gene polyadenylation signal (SV40 nucleotides 2770-2533). The BamHI to BglI fragment is derived from plasmid pBR322 ›4! (nucleotides 275-2422) but with an additional deletion between the SalI and AvaI sites (nucleotides 651-1425) following addition of a SalI linker to the AvaI site. The sequence from the BglI site to the XmnI site is from the .beta.-lactamase gene of plasmid pSP64 ›3!. Plasmid pEE6gpt contains the transcription unit encoding xanthine-guanine phosphoribosyl transferase (gpt) from plasmid pSV2gpt ›5! cloned into plasmid pEE6 as a BamHI fragment by the addition of a BamHI linker to the single PvuII site of plasmid pSV2gpt. By similar means, a derivative of plasmid PCMGS containing the transcription cassette for the xanthine-guanine phosphoribosyl transferase (gpt) gene from pEE6gpt was produced. The plasmid thus produced was labelled pCMGSgpt. Plasmid pEE6hCMV contains the hCMV-MIE promoter-enhancer and complete 5' untranslated sequence inserted by means of oligonucleotide linkers into the HindIII site of plasmid pEE6. Plasmid pEE6hCMVBglII is a derivative of pEE6hCMV in which the HindIII site upstream of the hCMV enhancer has been converted to a BglII site by blunt-ending and addition of a synthetic oligonucleotide linker. Plasmid pEE6HCLCBg is a vector derived from pEE6hCMV containing a coding sequence for a mouse-human chimeric Ig light chain from the B72.3 antibody ›6! inserted into the EcoRI site of pEE6hCMV such that the light chain is under the control of the hCMV-MIE promoter-enhancer. (The upstream HindIII site has also been converted to a BglII site by standard methods.) The 2.1 kb BamHI fragment from pSV2BamGS was inserted into pEE6HCLCBg to produce a plasmid pcLc2GS in which the Ig light chain and GS genes are transcribed in the same orientation with the GS gene downstream of the light chain gene. pEE6HCHHCL is a vector which contains sequences coding for both the heavy and light chains of the chimeric B72.3 antibody ›6! under the control of hCMV-MIE promoter enhancers. The 2.1 kb BamHI fragment from pSV2BamGS was inserted into pEE6HCHHCL to produce a plasmid pAb2GS in which the heavy and light chain genes and the GS gene are all transcribed in the same orientation in the order heavy chain, light chain, GS. A 3.1 kb BglII-BamHI fragment from pEE6HcLcBg was inserted into the BamHI site of pSV2GS to produce a plasmid pSV2GScLc in which the chimeric light chain gene and the GS gene are transcribed in the same orientation with the GS gene upstream of the light chain gene. Similarly, the 3.1 kb BglII-BamHI fragment of PEE6HCLCBg was inserted into the BamHI site of pCMGS to produce a plasmid pCMGS.CLC in which both genes are again in the same orientation. pEE6CHCBg is a plasmid containing the heavy chain gene of chimeric B72.3 antibody ›6! under the control of the hCNV-MIE promoter-enhancer and SV40 polyadenylation signal. The hCMV-MIE chain termination unit was excised from the plasmid as a 4.7 kb partial HindIII-BamHI fragment and inserted, by means of a HindIII-BamHI oligonucleotide adaptor, at the single BamHI site of psv2GScLc to form pSV2GScLccHc. The BamHI site upstream of the hCMV-MIE-cH chain transcription unit in pSV2GScLccHc was then removed by partial BamHI digestion, filling in with DNA polymerase I and religating to form pST6. A gene coding for a novel fibrinolytic enzyme of 90 kD molecular weight was isolated as a 2.8 kb HindIII to BglII fragment. This was then inserted between the HindIII and BclI sites of the expression plasmid pEE6hCMVBglII in the appropriate orientation such that the hCMV promoter directed transcription of the inserted gene. An SV40 Early-GS transcription unit was excised as a BamHI fragment from pSV2GS and inserted into the BglII site at the 5' end of the hCMV sequence in pEE6hCMVBglII, in the appropriate orientation such that transcription from the hCMV promoter and the SV40 early promoter is in the same direction. This formed the plasmid pEE690KGS. Cell Lines In the Examples, the following cell lines were used: NSO and P3-X63Ag8.653, which are non-producing variants of the mouse P3 mouse plasmacytoma line; Sp2/0, which is a non-producing mouse hybridoma cell line; and YB2/0, which is a non-producing rat hybridoma cell line. Media All cells were grown in either non-selective medium, Dulbecco's Minimum Essential Medium (DMEM) containing 2 mM glutamine, 100 .mu.M non-essential amino acids, 10% foetal calf serum and streptomycin/penicillin, or in glutamine-free DMX (G-DMEM) containing 500 .mu.m each of glutamate and asparagine, 30 .mu.M each of adenosine, quanosine, cytidine and uridine, 10 .mu.M thymidine, 100 .mu.M non-essential amino acids, 10% dialysed foetal calf serum and streptomycin/penicillin, or in derivatives of G-DMEM lacking various of these additives. Alternatively, cells were cultured in gpt-selective media, made using the following filter-sterilised stock solutions: 1) 50.times. each of hypoxanthine and thymidine; 2) 50.times. xanthine (12.5 mg/ml in 0.2M NaOH); 3) mycophenolic acid (MPA, 250 .mu.g/ml in 0.1M NaOH); and 4) 1M HCl. gpt-selective medium is made by mixing 93 ml of non-selective medium (described above), 2 ml solution 1), 2 ml solution 3) and 0.6 ml solution 4). 2.times. gpt is made by mixing 86 ml of non-selective medium with twice the above quantities of solutions 1) to 4). Linearisation of Plasmids In order to introduce them into cells all plasmids were linearised by digestion with an appropriate restriction enzyme which cuts at a single site in the plasmid and hence does not interfere with transcription of the relevant genes in mammalian cells. Typically 40 .mu.g of circular plasmid was digested in a volume of 400 .mu.l restriction buffer. The enzymes used for linearisation of the plasmids are shown in Table 1. TABLE 1 ______________________________________ Enzymes used for Linearisation of plasmid Plasmid Restriction Enzyme ______________________________________ PSVLGS.1 PvuI pSV2.GS PvuI pSV2.Bam GS PvuI pCMGS PvuI pCMGS.gpt PvuI PEE6.gpt SalI pcLc2GS SalI pAb2GS SalI pSV2.GSCLc TthIII pCMGS.cLc TthIII pST6 BamHI pEE690KGS SalI ______________________________________ Electroporation of Cells Cells were harvested while growing exponentially, washed once in phosphate-buffered saline (PBS) by centrifugation at 1200 rpm in a bench centrifuge and resuspended at a density of 10.sup.7 cells/ml in fresh ice cold PBS. One ml of cell suspension was added to the digested plasmid DNA (0.4 ml in restriction buffer) and incubated on ice for 5-10 minutes. The cell-DNA mixture was then subjected to 2 pulses of 2000 volts between aluminium electrodes spaced approximately 1 cm apart using a conventional electroporation apparatus having a capacitance of 14 .mu.F. Cells were then returned to ice for 5-10 minutes, resuspended in non-selective growth medium (DMEM) and distributed among 24-well culture trays. Selective medium (G-DMEM) was added subsequently as described below. |
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