Combinatorial gene expression using multiple episomal vectors

Episomal Vectors offer a powerful alternative to integrative recombination for transgene expression in mammalian cells. In this study, various combina tions of G protein-coupled receptors (GPCRs) and the G protein subunit G(i2 )alpha, were stably expressed from separate episomal vectors in 293-EBNA (2 93E) cells. Each episome did not adversely affect the others, as gauged by episomal copy number, steady-state mRNA levels and the presence of function al receptors and G protein. Cell lines expressing genes from multiple auton omously replicating Vectors were stable just two weeks after transfection, and remained stable in continuous culture for at least 5 months. Go-express ion of supplementary G(i2)alpha with receptor amplifies the magnitude of si gnal transduction thereby permitting the development of more sensitive high throughput functional assays. Given these results, combinatorial transfect ion is the strategy of choice for generating stable cell lines expressing m ultiple genes for the study of signal-transduction pathways or the evaluati on of receptor ligands. (C) 2000 Elsevier Science B.V. All rights reserved.