Gene targeting in human somatic cells. Complete inactivation of an interferon-inducible gene.
The role, if any, of the human interferon-inducible 6-16 gene in the establishment of a cellular antiviral state is unknown. To address this problem, and as part of a wider investigation of homologous recombination (HR) and its applications in somatic cells, we have been using HR to disrupt the 6-16 gene in human cell lines [Itzhaki, J. E. & Porter, A. C. G. (1991) Nucleic Acids Res. 19, 3835-3842.] We describe here the design and use of insertion and replacement-type targeting constructs based on a promoterless bacterial gpt gene that is activated by HR with the 6-16 gene. In HeLa cells, both targeting constructs underwent extrachromosomal HR with a cotransfected plasmid carrying the 6-16 gene. In a previously targeted clone derived from the fibrosarcoma cell line HT1080, the replacement construct underwent HR with either the modified or the unmodified 6-16 allele. The latter events generated doubly disrupted (6-16-/-) clones that failed to express any detectable 6-16 messenger RNA in response to interferon. Plaque assays of infected 6-16-/- cells showed that expression of the 6-16 gene was not required for the induction by interferon of an antiviral state against encephalomyocarditis virus, semliki forest virus or cocal virus.
Porter
A C
AC
Department of Biochemistry, Oxford University, England.
Itzhaki
J E
JE
D001483:Base Sequence; D017931:DNA Primers; D005786:Gene Expression Regulation; D020022:Genetic Predisposition to Disease; D006367:HeLa Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007372:Interferons / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010850:Picornaviridae Infections / Q000276:immunology; D011995:Recombination, Genetic; D018353:Rhabdoviridae Infections / Q000276:immunology; D014036:Togaviridae Infections / Q000276:immunology
D017931:DNA Primers; D007372:Interferons; D007041:Hypoxanthine Phosphoribosyltransferase