TARGETED DISRUPTION OF AN ESSENTIAL VERTEBRATE GENE - ASF SF2 IS REQUIRED FOR CELL VIABILITY/

Alternative splicing factor/splicing factor 2 (ASF/SF2) is the prototy pe of a family of nuclear proteins highly conserved throughout metazoa , the SR (serine/arginine) proteins. Based largely on in vitro studies , SR proteins have been suggested to play important roles in constitut ive and alternative splicing of pre-mRNAs. Here we describe the develo pment of a genetic system employing the chicken B-cell line DT40 to st udy the function of ASF/SF2 in vivo. The high level of homologous reco mbination and rapid growth rate of these cells allowed us to show firs t that ASF/SF2 is an essential gene, and then to perform targeted disr uption of both ASF/SF2 alleles, by creating a cell line in which the o nly source of ASF/SF2 is a human cDNA controlled by a tetracycline (te t)-repressible promoter. We show that addition of tet to these cells r esults in rapid depletion of ASF/SE2, concomitant accumulation of inco mpletely processed pre-mRNA, and subsequent cell death. The tet-induce d lethality could be rescued by plasmids expressing wild-type ASF/SF2, but not several mutant derivatives, or other SR proteins. Heterozygou s cell lines overexpressing human ASF/SE2 displayed significant reduct ions of endogenous ASF/SF2 mRNA, suggesting that ASF/SF2 mRNA levels a re controlled by an autoregulatory loop. This system provides a novel method for genetic analysis of factors that function in basic processe s in vertebrate cells.