EXPRESSION CLONING OF ONCOGENES BY RETROVIRAL TRANSFER OF CDNA LIBRARIES

Citation
I. Whitehead et al., EXPRESSION CLONING OF ONCOGENES BY RETROVIRAL TRANSFER OF CDNA LIBRARIES, Molecular and cellular biology, 15(2), 1995, pp. 704-710
Citations number
52
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0270-7306
Volume
15
Issue
2
Year of publication
1995
Pages
704 - 710
Database
ISI
SICI code
0270-7306(1995)15:2<704:ECOOBR>2.0.ZU;2-7
Abstract
A cDNA library transfer system based on retroviral vectors has been de veloped for expression cloning in mammalian cells, The use of retrovir al vectors results in stable cDNA transfer efficiencies which are at l east 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 N IH 3T3 fibroblasts, as measured by loss of contact inhibition of proli feration Nineteen different transforming cDNAs were isolated from a to tal of 300,000 transferred cDNA clones. Three of these cDNAs were deri ved from known oncogenes (raf-1, lck, and ect2), while nine others wer e derived from genes which had been cloned previously but were not kno wn to have the ability to transform fibroblasts (beta-catenin, thrombi n receptor, phospholipase C-gamma(2) and Spi-2 protease inhibitor gene s). The Spi-2 cDNA was expressed in antisense orientation and therefor e is likely to act as an inhibitor of an endogenous transformation sup pressor. Seven novel cDNAs with transforming activities, including tho se for three new members of the CDC24 family of guanine nucleotide exc hange factors, were also cloned from the retroviral cDNA libraries, Re troviral transfer of libraries should be generally useful for cloning cDNAs,which confer selectable phenotypes on many different types of ma mmalian cells.