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Survival, mutagenesis, and host cell reactivation in a Chinese hamster ovary cell ERCC1 knock-out mutant
R L Rolig1, S K Layher, B Santi
1Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957, USA.
Abstract:
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.
Insights
Gene targeting disrupted the ERCC1 gene in CHO-K1 cells, creating a severe mutagen sensitivity phenotype. This study clarifies ERCC1 gene function in DNA repair and highlights differences in Chinese hamster ovary cell lines.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- The ERCC1 gene is crucial for nucleotide excision repair (NER), a major DNA repair pathway.
- Understanding ERCC1 function is vital for comprehending cellular responses to DNA damage.
Purpose of the Study:
- To investigate the role of the ERCC1 gene in DNA repair and mutagenesis using gene targeting in CHO-K1 cells.
- To characterize the DNA repair and sensitivity phenotypes of an ERCC1-disrupted Chinese hamster ovary cell line.
Main Methods:
- Gene targeting via positive-negative selection to disrupt the ERCC1 gene in CHO-K1 cells.
- Southern and Northern blot analyses to confirm gene disruption and absence of ERCC1 mRNA.
- Assessment of cellular sensitivity to UV radiation and mitomycin C.
- Mutation frequency analysis at the HPRT locus and host cell reactivation (HCR) assays.
Main Results:
- A CHO-K1 cell clone (CHO-7-27) with targeted disruption of ERCC1 exon V and no ERCC1 mRNA expression was generated.
- CHO-7-27 cells exhibited severe sensitivity to UV and mitomycin C, and were UV-hypermutable.
- The ERCC1-disrupted cells showed a severely deficient host cell reactivation (HCR) phenotype for UV-damaged viral DNA.
- Chinese hamster ovary (CHO)-K1 cells are hemizygous for the ERCC1 gene.
Conclusions:
- Targeted disruption of ERCC1 in CHO-K1 cells leads to severe DNA repair deficiencies and hypersensitivity to mutagens.
- The observed phenotypes in CHO-7-27 cells are consistent with a critical role for ERCC1 in NER.
- The study elucidates that differences in ERCC1 mutant phenotypes between CHO-K1 and CHO-AA8 cell lines are not due to intrinsic genetic variations between the parental lines.