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.
Whitehead
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Department of Medical Genetics, University of British Columbia, Canada.
Kirk
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H
Kay
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R
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003598:Cytoskeletal Proteins / Q000096:biosynthesis; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009857:Oncogenes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019908:Proto-Oncogene Proteins c-raf; D018179:Receptors, Thrombin / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012190:Retroviridae; D015843:Serpins / Q000096:biosynthesis; D015534:Trans-Activators; D014162:Transfection; D010738:Type C Phospholipases / Q000096:biosynthesis; D014764:Viral Proteins; D051176:beta Catenin
C495267:CTNNB1 protein, mouse; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D018076:DNA, Complementary; C080509:Ect2 protein, mouse; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D015843:Serpins; D015534:Trans-Activators; D014764:Viral Proteins; D051176:beta Catenin; C074641:interleukin-1beta-converting enzyme inhibitor; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D010738:Type C Phospholipases