Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.
Alternative splicing factor/splicing factor 2 (ASF/SF2) is the prototype 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 constitutive and alternative splicing of pre-mRNAs. Here we describe the development of a genetic system employing the chicken B-cell line DT40 to study the function of ASF/SF2 in vivo. The high level of homologous recombination and rapid growth rate of these cells allowed us to show first that ASF/SF2 is an essential gene, and then to perform targeted disruption of both ASF/SF2 alleles, by creating a cell line in which the only source of ASF/SF2 is a human cDNA controlled by a tetracycline (tet)-repressible promoter. We show that addition of tet to these cells results in rapid depletion of ASF/SF2, concomitant accumulation of incompletely processed pre-mRNA, and subsequent cell death. The tet-induced lethality could be rescued by plasmids expressing wild-type ASF/SF2, but not several mutant derivatives, or other SR proteins. Heterozygous cell lines overexpressing human ASF/SF2 displayed significant reductions of endogenous ASF/SF2 mRNA, suggesting that ASF/SF2 mRNA levels are controlled by an autoregulatory loop. This system provides a novel method for genetic analysis of factors that function in basic processes in vertebrate cells.
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
J
J
Wang
Y
Y
Takagaki
J L
JL
Manley
D000483:Alleles; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism; D016923:Cell Death; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D002645:Chickens; D005786:Gene Expression Regulation; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics*; D012322:RNA Precursors; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D013752:Tetracycline / Q000494:pharmacology; D014714:Vertebrates / Q000235:genetics
D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D013752:Tetracycline