Genetic screens in mammalian cells by enhanced retroviral mutagens.
Genetic approaches such as retrovirus-mediated mutagenesis and cDNA expression libraries have contributed greatly to our understanding of signal transduction in mammalian cells. However, previously described methods for retroviral insertional mutagenesis are hindered by low mutagenesis rates and difficulties in cloning mutated genes. cDNA expression library methods are usually cell-type dependent and bias towards abundant and short messages. With the near completion of the genome projects, alternative genetic methods are needed where large numbers of genes can be more easily isolated and biochemically studied. We have developed a novel retrovirus-mediated genetic screening method in cultured cells. To achieve efficient and regulated mutagenesis, we constructed Enhanced Retroviral Mutagen (ERM) vectors that contained several engineered sequences (e.g., an ERM Tag and a splice donor) controlled by a tetracycline-responsive promoter. Endogenous genes can thus be randomly activated and tagged in a conditional system. NIH3T3 cells were used to screen for focus-forming genes using the ERM strategy. We showed that these added sequences increased the screening efficiency by >10-fold, and allowed more direct identification of the genes targeted. Sequence analysis of approximately 10% of the >600 focus clones recovered revealed both known oncogenes and novel factors such as protein kinases and GTP/GDP exchange proteins. The ERM strategy should help to facilitate large-scale gene identification in diverse pathways and integrate both genetic (with the completion of the genome projects) and functional information more readily.
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Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Yang
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Yang
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Songyang
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D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering; D005820:Genetic Testing / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D016254:Mutagenesis, Insertional / Q000235:genetics*; D019814:Myristic Acid / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance; D014170:Transformation, Genetic / Q000235:genetics; D016662:Virus Integration / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D013752:Tetracycline