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. 1995 Feb;15(2):704–710. doi: 10.1128/mcb.15.2.704

Expression cloning of oncogenes by retroviral transfer of cDNA libraries.

I Whitehead 1, H Kirk 1, R Kay 1
PMCID: PMC231935  PMID: 7823939

Abstract

a cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (beta-catenin, thrombin receptor, phospholipase C-gamma 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.

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Selected References

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  1. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  2. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernard H. U., Krämmer G., Röwekamp W. G. Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture. Exp Cell Res. 1985 May;158(1):237–243. doi: 10.1016/0014-4827(85)90446-x. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bishop J. M. Molecular themes in oncogenesis. Cell. 1991 Jan 25;64(2):235–248. doi: 10.1016/0092-8674(91)90636-d. [DOI] [PubMed] [Google Scholar]
  6. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  7. Brown B. A., Padgett R. W., Hardies S. C., Hutchison C. A., 3rd, Edgell M. H. beta-globin transcript found in induced murine erythroleukemia cells is homologous to the beta h0 and beta h1 genes. Proc Natl Acad Sci U S A. 1982 May;79(9):2753–2757. doi: 10.1073/pnas.79.9.2753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan A. M., Fleming T. P., McGovern E. S., Chedid M., Miki T., Aaronson S. A. Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product. Mol Cell Biol. 1993 Feb;13(2):762–768. doi: 10.1128/mcb.13.2.762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen C. A., Okayama H. Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques. 1988 Jul-Aug;6(7):632–638. [PubMed] [Google Scholar]
  10. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Cooper C. S., Park M., Blair D. G., Tainsky M. A., Huebner K., Croce C. M., Vande Woude G. F. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature. 1984 Sep 6;311(5981):29–33. doi: 10.1038/311029a0. [DOI] [PubMed] [Google Scholar]
  13. Emori Y., Homma Y., Sorimachi H., Kawasaki H., Nakanishi O., Suzuki K., Takenawa T. A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity. J Biol Chem. 1989 Dec 25;264(36):21885–21890. [PubMed] [Google Scholar]
  14. Eva A., Aaronson S. A. Isolation of a new human oncogene from a diffuse B-cell lymphoma. Nature. 1985 Jul 18;316(6025):273–275. doi: 10.1038/316273a0. [DOI] [PubMed] [Google Scholar]
  15. Gorman C. M., Howard B. H., Reeves R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 1983 Nov 11;11(21):7631–7648. doi: 10.1093/nar/11.21.7631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Greenberger J. S., Sakakeeny M. A., Humphries R. K., Eaves C. J., Eckner R. J. Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines. Proc Natl Acad Sci U S A. 1983 May;80(10):2931–2935. doi: 10.1073/pnas.80.10.2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gutkind J. S., Novotny E. A., Brann M. R., Robbins K. C. Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4703–4707. doi: 10.1073/pnas.88.11.4703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hamaguchi M., Matsuyoshi N., Ohnishi Y., Gotoh B., Takeichi M., Nagai Y. p60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin-catenin cell adhesion system. EMBO J. 1993 Jan;12(1):307–314. doi: 10.1002/j.1460-2075.1993.tb05658.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inglis J. D., Lee M., Davidson D. R., Hill R. E. Isolation of two cDNAs encoding novel alpha 1-antichymotrypsin-like proteins in a murine chondrocytic cell line. Gene. 1991 Oct 15;106(2):213–220. doi: 10.1016/0378-1119(91)90201-l. [DOI] [PubMed] [Google Scholar]
  20. Jacobson A. Purification and fractionation of poly(A)+ RNA. Methods Enzymol. 1987;152:254–261. doi: 10.1016/0076-6879(87)52028-6. [DOI] [PubMed] [Google Scholar]
  21. Johnson G. R., Gonda T. J., Metcalf D., Hariharan I. K., Cory S. A lethal myeloproliferative syndrome in mice transplanted with bone marrow cells infected with a retrovirus expressing granulocyte-macrophage colony stimulating factor. EMBO J. 1989 Feb;8(2):441–448. doi: 10.1002/j.1460-2075.1989.tb03396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Julius D., Livelli T. J., Jessell T. M., Axel R. Ectopic expression of the serotonin 1c receptor and the triggering of malignant transformation. Science. 1989 Jun 2;244(4908):1057–1062. doi: 10.1126/science.2727693. [DOI] [PubMed] [Google Scholar]
  23. Kay R., Rosten P. M., Humphries R. K. CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor. J Immunol. 1991 Aug 15;147(4):1412–1416. [PubMed] [Google Scholar]
  24. Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem. 1991 Oct 25;266(30):19867–19870. [PubMed] [Google Scholar]
  25. LaMorte V. J., Kennedy E. D., Collins L. R., Goldstein D., Harootunian A. T., Brown J. H., Feramisco J. R. A requirement for Ras protein function in thrombin-stimulated mitogenesis in astrocytoma cells. J Biol Chem. 1993 Sep 15;268(26):19411–19415. [PubMed] [Google Scholar]
  26. Li P., Wood K., Mamon H., Haser W., Roberts T. Raf-1: a kinase currently without a cause but not lacking in effects. Cell. 1991 Feb 8;64(3):479–482. doi: 10.1016/0092-8674(91)90228-q. [DOI] [PubMed] [Google Scholar]
  27. Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marth J. D., Cooper J. A., King C. S., Ziegler S. F., Tinker D. A., Overell R. W., Krebs E. G., Perlmutter R. M. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). Mol Cell Biol. 1988 Feb;8(2):540–550. doi: 10.1128/mcb.8.2.540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Martin-Zanca D., Hughes S. H., Barbacid M. A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences. 1986 Feb 27-Mar 5Nature. 319(6056):743–748. doi: 10.1038/319743a0. [DOI] [PubMed] [Google Scholar]
  30. Miki T., Fleming T. P., Bottaro D. P., Rubin J. S., Ron D., Aaronson S. A. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science. 1991 Jan 4;251(4989):72–75. doi: 10.1126/science.1846048. [DOI] [PubMed] [Google Scholar]
  31. Miki T., Fleming T. P., Crescenzi M., Molloy C. J., Blam S. B., Reynolds S. H., Aaronson S. A. Development of a highly efficient expression cDNA cloning system: application to oncogene isolation. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5167–5171. doi: 10.1073/pnas.88.12.5167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miki T., Smith C. L., Long J. E., Eva A., Fleming T. P. Oncogene ect2 is related to regulators of small GTP-binding proteins. Nature. 1993 Apr 1;362(6419):462–465. doi: 10.1038/362462a0. [DOI] [PubMed] [Google Scholar]
  33. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  34. Miyoshi J., Higashi T., Mukai H., Ohuchi T., Kakunaga T. Structure and transforming potential of the human cot oncogene encoding a putative protein kinase. Mol Cell Biol. 1991 Aug;11(8):4088–4096. doi: 10.1128/mcb.11.8.4088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mizushima S., Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990 Sep 11;18(17):5322–5322. doi: 10.1093/nar/18.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Murphy A. J., Efstratiadis A. Cloning vectors for expression of cDNA libraries in mammalian cells. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8277–8281. doi: 10.1073/pnas.84.23.8277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. O'Bryan J. P., Frye R. A., Cogswell P. C., Neubauer A., Kitch B., Prokop C., Espinosa R., 3rd, Le Beau M. M., Earp H. S., Liu E. T. axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol. 1991 Oct;11(10):5016–5031. doi: 10.1128/mcb.11.10.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pear W. S., Nolan G. P., Scott M. L., Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. doi: 10.1073/pnas.90.18.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pineau J. C., Vol S., Tichet J. Modélisation et standardisation de la corpulence chez les Français âgés de 18 à 59 ans. C R Acad Sci III. 1995 Jun;318(6):709–714. [PubMed] [Google Scholar]
  41. Rayner J. R., Gonda T. J. A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector. Mol Cell Biol. 1994 Feb;14(2):880–887. doi: 10.1128/mcb.14.2.880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Seed B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. 1987 Oct 29-Nov 4Nature. 329(6142):840–842. doi: 10.1038/329840a0. [DOI] [PubMed] [Google Scholar]
  43. Seed B., Aruffo A. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc Natl Acad Sci U S A. 1987 May;84(10):3365–3369. doi: 10.1073/pnas.84.10.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Seed B. Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. Nucleic Acids Res. 1983 Apr 25;11(8):2427–2445. doi: 10.1093/nar/11.8.2427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shimizu K., Goldfarb M., Perucho M., Wigler M. Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line. Proc Natl Acad Sci U S A. 1983 Jan;80(2):383–387. doi: 10.1073/pnas.80.2.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stanton V. P., Jr, Nichols D. W., Laudano A. P., Cooper G. M. Definition of the human raf amino-terminal regulatory region by deletion mutagenesis. Mol Cell Biol. 1989 Feb;9(2):639–647. doi: 10.1128/mcb.9.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Thomas K. R., Capecchi M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503–512. doi: 10.1016/0092-8674(87)90646-5. [DOI] [PubMed] [Google Scholar]
  48. Vogeli G., Horn E., Laurent M., Nath P. Recombinant DNA techniques: storage and screening of cDNA libraries with large numbers of individual colonies from initial transformations. Anal Biochem. 1985 Dec;151(2):442–444. doi: 10.1016/0003-2697(85)90202-7. [DOI] [PubMed] [Google Scholar]
  49. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]

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