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.[1]References
- Gene targeting in human somatic cells. Complete inactivation of an interferon-inducible gene. Porter, A.C., Itzhaki, J.E. Eur. J. Biochem. (1993) [Pubmed]
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