Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.
10.1101/gad.10.20.2588
Crossref journal-article
Cold Spring Harbor Laboratory
Genes & Development (246)
Abstract

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.

Bibliography

Wang, J., Takagaki, Y., & Manley, J. L. (1996). Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability. Genes & Development, 10(20), 2588–2599.

Authors 3
  1. J Wang (first)
  2. Y Takagaki (additional)
  3. J L Manley (additional)
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Dates
Type When
Created 18 years, 2 months ago (June 5, 2007, 5:15 p.m.)
Deposited 3 years, 9 months ago (Nov. 14, 2021, 9:54 p.m.)
Indexed 1 month, 1 week ago (July 14, 2025, 11:18 p.m.)
Issued 28 years, 10 months ago (Oct. 15, 1996)
Published 28 years, 10 months ago (Oct. 15, 1996)
Published Online 28 years, 10 months ago (Oct. 15, 1996)
Published Print 28 years, 10 months ago (Oct. 15, 1996)
Funders 0

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@article{Wang_1996, title={Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.}, volume={10}, ISSN={1549-5477}, url={http://dx.doi.org/10.1101/gad.10.20.2588}, DOI={10.1101/gad.10.20.2588}, number={20}, journal={Genes & Development}, publisher={Cold Spring Harbor Laboratory}, author={Wang, J and Takagaki, Y and Manley, J L}, year={1996}, month=oct, pages={2588–2599} }