Expression cloning of oncogenes by retroviral transfer of cDNA libraries

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.

[1]  J. Rayner,et al.  A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector. , 1994, Molecular and cellular biology.

[2]  Mark S. Boguski,et al.  Proteins regulating Ras and its relatives , 1993, Nature.

[3]  A. Harootunian,et al.  A requirement for Ras protein function in thrombin-stimulated mitogenesis in astrocytoma cells. , 1993, The Journal of biological chemistry.

[4]  S. Aaronson,et al.  Expression cDNA cloning of a serine kinase transforming gene. , 1993, Oncogene.

[5]  T. Fleming,et al.  Oncogene ect2 is related to regulators of small GTP-binding proteins , 1993, Nature.

[6]  T. Fleming,et al.  Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product , 1993, Molecular and cellular biology.

[7]  M. Takeichi,et al.  p60v‐src causes tyrosine phosphorylation and inactivation of the N‐cadherin‐catenin cell adhesion system. , 1993, The EMBO journal.

[8]  G. Nolan,et al.  Production of high-titer helper-free retroviruses by transient transfection. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Kozak.  Structural features in eukaryotic mRNAs that modulate the initiation of translation. , 1991, The Journal of biological chemistry.

[10]  D. Davidson,et al.  Isolation of two cDNAs encoding novel alpha 1-antichymotrypsin-like proteins in a murine chondrocytic cell line. , 1991, Gene.

[11]  R. Espinosa,et al.  axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase , 1991, Molecular and cellular biology.

[12]  R. Humphries,et al.  CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor. , 1991, Journal of immunology.

[13]  J. Miyoshi,et al.  Structure and transforming potential of the human cot oncogene encoding a putative protein kinase , 1991, Molecular and cellular biology.

[14]  T. Fleming,et al.  Development of a highly efficient expression cDNA cloning system: application to oncogene isolation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Brann,et al.  Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[16]  V. Wheaton,et al.  Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation , 1991, Cell.

[17]  W. Haser,et al.  Raf-1: A kinase currently without a cause but not lacking in effects , 1991, Cell.

[18]  J. Bishop.  Molecular themes in oncogenesis , 1991, Cell.

[19]  J. Rubin,et al.  Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. , 1991, Science.

[20]  S. Nagata,et al.  pEF-BOS, a powerful mammalian expression vector. , 1990, Nucleic acids research.

[21]  H. Land,et al.  Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. , 1990, Nucleic acids research.

[22]  H. Kawasaki,et al.  A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity. , 1989, The Journal of biological chemistry.

[23]  Miller Ad,et al.  Improved Retroviral Vectors for Gene Transfer and Expression , 1989 .

[24]  T. Jessell,et al.  Ectopic expression of the serotonin 1c receptor and the triggering of malignant transformation. , 1989, Science.

[25]  I. Hariharan,et al.  A lethal myeloproliferative syndrome in mice transplanted with bone marrow cells infected with a retrovirus expressing granulocyte‐macrophage colony stimulating factor. , 1989, The EMBO journal.

[26]  V. Stanton,et al.  Definition of the human raf amino-terminal regulatory region by deletion mutagenesis , 1989, Molecular and cellular biology.

[27]  A. Miller,et al.  Improved retroviral vectors for gene transfer and expression. , 1989, BioTechniques.

[28]  H. Okayama,et al.  Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. , 1988, BioTechniques.

[29]  S. Goff,et al.  A safe packaging line for gene transfer: separating viral genes on two different plasmids , 1988, Journal of virology.

[30]  Jonathan A. Cooper,et al.  Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck) , 1988, Molecular and cellular biology.

[31]  A. Efstratiadis,et al.  Cloning vectors for expression of cDNA libraries in mammalian cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Capecchi,et al.  Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells , 1987, Cell.

[33]  H. Okayama,et al.  High-efficiency transformation of mammalian cells by plasmid DNA. , 1987, Molecular and cellular biology.

[34]  B. Seed,et al.  Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[35]  M. Bender,et al.  Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region , 1987, Journal of virology.

[36]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[37]  A. Jacobson.  [25] Purification and fractionation of poly(A)+ RNA , 1987 .

[38]  A. Jacobson.  Purification and fractionation of poly(A)+ RNA. , 1987, Methods in enzymology.

[39]  B. Seed.  An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2 , 1987, Nature.

[40]  M. Barbacid,et al.  A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences , 1986, Nature.

[41]  P. Nath,et al.  Recombinant DNA techniques: storage and screening of cDNA libraries with large numbers of individual colonies from initial transformations. , 1985, Analytical biochemistry.

[42]  S. Aaronson,et al.  Isolation of a new human oncogene from a diffuse B-cell lymphoma , 1985, Nature.

[43]  G. Krämmer,et al.  Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture. , 1985, Experimental cell research.

[44]  C. Cooper,et al.  Molecular cloning of a new transforming gene from a chemically transformed human cell line , 1984, Nature.

[45]  B. Howard,et al.  Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. , 1983, Nucleic acids research.

[46]  J. Greenberger,et al.  Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[47]  B. Seed.  Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. , 1983, Nucleic acids research.

[48]  M. Wigler,et al.  Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[49]  B. Reiss,et al.  Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. , 1982, Gene.

[50]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.