Gene targeting in human somatic cells
PORTER ACG., ITZHAKI JE.
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.