Generation of mismatch repair-profi cient cell lines from human HCT116 cells

We have used HCT116 as a parental cell line for targeted disruption of DNA repair related genes, because HCT116 has many advantages for gene-disruption studies (such as near dipoloid karyotype, stable chromosome number).However, HCT116 has a disadvantage that is the defect in mismatch repair (MMR) due to a point mutation (from serine [TCA] to stop [TAA] at amino acid residue 252) in both allele of one of MMR genes, hMLH1. In order to rescue the MMR-defect in HCT116 cells, we tried to replace the mutated portion of hMLH1 gene with normal hMLH1 fragment using the gene-replacement vector constructed with the normal hMLH1 fragment. The vector also has neo gene as a dominant selective marker. After introducing the vector DNA into HCT116 cells by electroporation, we selected G418-resistant clones. Out of these clones we selected the clones in which mutated portion of hMLH1 were replaced with normal sequence examining by PCR and Southern blotting.Furthermore normal size of hMLH1 protein was detected in these clones.Because MMR-deficient cells have been reported to show 10 times more resistance to the drug, 6-thioguanine (6TG), and also show more than 100 times higher spontaneous mutation frequency, we examined the hMLH1-proficient cells for the sensitivity to 6TG and spontaneous mutation frequencies at HGPRT locus. These cells showed normal sensitivity to 6TG and normal levels of spontaneous mutation frequency indicating that these cells were normal in MMR function.The 32nd Annual Meeting of the Molecular Biology Society of Japa