Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Jan;4(1):166–172. doi: 10.1128/mcb.4.1.166

Inducible expression of amplified human beta interferon genes in CHO cells.

F McCormick, M Trahey, M Innis, B Dieckmann, G Ringold
PMCID: PMC368671  PMID: 6700582

Abstract

Plasmid DNA containing the human beta-interferon (IFN-beta) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-beta after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-beta gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 10(10) U/liter medium in culture supernatants. Constitutive production of IFN-beta rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-beta had acquired resistance to cytotoxic antiviral effects of IFN-beta. Two forms of human IFN-beta were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-beta production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-beta production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-beta-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-beta.

Full text

PDF
171

Images in this article

168Image
on p.168
169Image
on p.169
170Image
on p.170

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bose S., Gurari-Rotman D., Ruegg U. T., Corley L., Anfinsen C. B. Apparent dispensability of the carbohydrate moiety of human interferon for antiviral activity. J Biol Chem. 1976 Mar 25;251(6):1659–1662. [PubMed] [Google Scholar]
  2. Canaani D., Berg P. Regulated expression of human interferon beta 1 gene after transduction into cultured mouse and rabbit cells. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5166–5170. doi: 10.1073/pnas.79.17.5166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chapman A. B., Costello M. A., Lee F., Ringold G. M. Amplification and hormone-regulated expression of a mouse mammary tumor virus-Eco gpt fusion plasmid in mouse 3T6 cells. Mol Cell Biol. 1983 Aug;3(8):1421–1429. doi: 10.1128/mcb.3.8.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  5. Derynck R., Content J., DeClercq E., Volckaert G., Tavernier J., Devos R., Fiers W. Isolation and structure of a human fibroblast interferon gene. Nature. 1980 Jun 19;285(5766):542–547. doi: 10.1038/285542a0. [DOI] [PubMed] [Google Scholar]
  6. Goeddel D. V., Shepard H. M., Yelverton E., Leung D., Crea R., Sloma A., Pestka S. Synthesis of human fibroblast interferon by E. coli. Nucleic Acids Res. 1980 Sep 25;8(18):4057–4074. doi: 10.1093/nar/8.18.4057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goeddel D. V., Yelverton E., Ullrich A., Heyneker H. L., Miozzari G., Holmes W., Seeburg P. H., Dull T., May L., Stebbing N. Human leukocyte interferon produced by E. coli is biologically active. Nature. 1980 Oct 2;287(5781):411–416. doi: 10.1038/287411a0. [DOI] [PubMed] [Google Scholar]
  8. Gray P. W., Leung D. W., Pennica D., Yelverton E., Najarian R., Simonsen C. C., Derynck R., Sherwood P. J., Wallace D. M., Berger S. L. Expression of human immune interferon cDNA in E. coli and monkey cells. Nature. 1982 Feb 11;295(5849):503–508. doi: 10.1038/295503a0. [DOI] [PubMed] [Google Scholar]
  9. Hauser H., Gross G., Bruns W., Hochkeppel H. K., Mayr U., Collins J. Inducibility of human beta-interferon gene in mouse L-cell clones. Nature. 1982 Jun 24;297(5868):650–654. doi: 10.1038/297650a0. [DOI] [PubMed] [Google Scholar]
  10. Havell E. A., Vilcek J. Production of high-titered interferon in cultures of human diploid cells. Antimicrob Agents Chemother. 1972 Dec;2(6):476–484. doi: 10.1128/aac.2.6.476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Houghton M., Stewart A. G., Doel S. M., Emtage J. S., Eaton M. A., Smith J. C., Patel T. P., Lewis H. M., Porter A. G., Birch J. R. The amino-terminal sequence of human fibroblast interferon as deduced from reverse transcripts obtained using synthetic oligonucleotide primers. Nucleic Acids Res. 1980 May 10;8(9):1913–1931. doi: 10.1093/nar/8.9.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hu N., Messing J. The making of strand-specific M13 probes. Gene. 1982 Mar;17(3):271–277. doi: 10.1016/0378-1119(82)90143-3. [DOI] [PubMed] [Google Scholar]
  13. Lee F., Mulligan R., Berg P., Ringold G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228–232. doi: 10.1038/294228a0. [DOI] [PubMed] [Google Scholar]
  14. Maeda S., McCandliss R., Gross M., Sloma A., Familletti P. C., Tabor J. M., Evinger M., Levy W. P., Pestka S. Construction and identification of bacterial plasmids containing nucleotide sequence for human leukocyte interferon. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7010–7013. doi: 10.1073/pnas.77.12.7010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mitrani-Rosenbaum S., Maroteaux L., Mory Y., Revel M., Howley P. M. Inducible expression of the human interferon beta 1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells. Mol Cell Biol. 1983 Feb;3(2):233–240. doi: 10.1128/mcb.3.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nagata S., Taira H., Hall A., Johnsrud L., Streuli M., Ecsödi J., Boll W., Cantell K., Weissmann C. Synthesis in E. coli of a polypeptide with human leukocyte interferon activity. Nature. 1980 Mar 27;284(5754):316–320. doi: 10.1038/284316a0. [DOI] [PubMed] [Google Scholar]
  17. Ohno S., Taniguchi T. Inducer-responsive expression of the cloned human interferon beta 1 gene introduced into cultured mouse cells. Nucleic Acids Res. 1982 Feb 11;10(3):967–977. doi: 10.1093/nar/10.3.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pestka S., Baron S. Definition and classification of the interferons. Methods Enzymol. 1981;78(Pt A):3–14. doi: 10.1016/0076-6879(81)78091-1. [DOI] [PubMed] [Google Scholar]
  19. Pitha P. M., Ciufo D. M., Kellum M., Raj N. B., Reyes G. R., Hayward G. S. Induction of human beta-interferon synthesis with poly(rI . rC) in mouse cells transfected with cloned cDNA plasmids. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4337–4341. doi: 10.1073/pnas.79.14.4337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  21. Ringold G., Dieckmann B., Lee F. Co-expression and amplification of dihydrofolate reductase cDNA and the Escherichia coli XGPRT gene in Chinese hamster ovary cells. J Mol Appl Genet. 1981;1(3):165–175. [PubMed] [Google Scholar]
  22. Schimke R. T., Kaufman R. J., Alt F. W., Kellems R. F. Gene amplification and drug resistance in cultured murine cells. Science. 1978 Dec 8;202(4372):1051–1055. doi: 10.1126/science.715457. [DOI] [PubMed] [Google Scholar]
  23. Smith A. E., Smith R., Paucha E. Extraction and fingerprint analysis of simian virus 40 large and small T-antigens. J Virol. 1978 Oct;28(1):140–153. doi: 10.1128/jvi.28.1.140-153.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stetler D., Das H., Nunberg J. H., Saiki R., Sheng-Dong R., Mullis K. B., Weissman S. M., Erlich H. A. Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5966–5970. doi: 10.1073/pnas.79.19.5966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tavernier J., Gheysen D., Duerinck F., Van der Heyden J., Fiers W. Deletion mapping of the inducible promoter of human IFN-beta gene. Nature. 1983 Feb 17;301(5901):634–636. doi: 10.1038/301634a0. [DOI] [PubMed] [Google Scholar]
  26. Urlaub G., Chasin L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yip Y. K., Vilcek J. Production of human fibroblast interferon in the presence of the glycosylation inhibitor tunicamycin. Methods Enzymol. 1981;78(Pt A):212–219. doi: 10.1016/0076-6879(81)78120-5. [DOI] [PubMed] [Google Scholar]
  28. Zinn K., Mellon P., Ptashne M., Maniatis T. Regulated expression of an extrachromosomal human beta-interferon gene in mouse cells. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4897–4901. doi: 10.1073/pnas.79.16.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES