Survival, mutagenesis, and host cell reactivation in a Chinese hamster ovary cell ERCC1 knock-out mutant
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Positive selection-negative selection gene targeting was used to disrupt the nucleotide excision repair gene ERCC1 in a Chinese hamster ovary cell line, CHO-K1. Southern and Northern analysis showed that a cell clone isolated by this targeting approach, CHO-7-27, had an ERCC1 gene structure consistent with targeted disruption of ERCC1 exon V, and did not express ERCC1 mRNA. CHO-7-27 was further characterized with respect to UV and mitomycin C sensitivities, and was shown to exhibit severe mutagen sensitivity phenotypes consistent with those of other CHO cell ERCC1 mutants. Mutation frequency experiments showed that CHO-7-27 was UV-hypermutable at the hypoxanthine-guanine phosphoribosyltransferase locus. Experiments assessing host cell reactivation of viral DNA synthesis for UV-irradiated adenovirus showed that CHO7-27 exhibited a severely deficient HCR phenotype similar to that of UV20 cells. Our results demonstrate that CHOK1 cells are hemizygous for the ERCC1 gene, and show that the comparatively mild mutagen sensitivities and lack of severely deficient HCR phenotypes of conventionally derived CHO-K1 ERCC1 mutants, in contrast to the severe phenotypes of CHO-AA8-derived mutants, are not due to any intrinsic genetic differences between CHO-K1 and CHO-AA8 parental cell lines.
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