Isolation of oncogenes by expression cDNA cloning

This chapter discusses the isolation of oncogenes by expression cDNA cloning. By means of genomic DNA transfection-transformation assays utilizing NIH/3T3 mouse fibroblasts as recipient cells, various oncogenes have been isolated from both human and rodent tumors. In the cDNA form, most genes are sufficiently small to achieve efficient transfer and are certain to be expressed when a strong constitutive promoter in the vector is used. Many mammalian expression cloning systems are based on transient high levels of expression driven by the simian virus 40 (SV40) promoter in COS cells. However, selection of cells showing certain biologic phenotypes may only be practical by means of stable expression. In order for a single cDNA to register a stable phenotype, it is necessary to develop a system capable of efficient synthesis of full-length cDNAs, unidirectional insertion of cDNA fragments into expression vectors, and a high level of gene expression. Moreover, a method to rescue integrated cDNA clones from mammalian cells is also required. The chapter presents an efficient cDNA cloning system using stable expression. It also describes the steps that are needed to isolate transforming cDNAs using this system.