SURVIVAL, MUTAGENESIS, AND HOST-CELL REACTIVATION IN A CHINESE-HAMSTER OVARY CELL ERCC1 KNOCK-OUT MUTANT

Positive selection-negative selection gene targeting was used to disru pt the nucleotide excision repair gene ERCC1 in a Chinese hamster ovar y cell line, CHO-K1. Southern and Northern analysis showed that a cell clone isolated by this targeting approach, CHO-7-27, had an ERCC1 gen e structure consistent with targeted disruption of ERCC1 exon V, and d id not express ERCC1 mRNA. CHO-7-27 was further characterized with res pect to UV and mitomycin C sensitivities, and was shown to exhibit sev ere mutagen sensitivity phenotypes consistent with those of other CHO cell ERCC1 mutants, Mutation frequency experiments showed that CHO-7-2 7 was UV-hypermutable at the hypoxanthine-guanine phosphoribosyltransf erase 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-AAS-derived mutants, are not due to any intrinsic genetic differences between CHO -K1 and CHO-AA8 parental cell lines.