Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations

We have previously described the use of homologous recombination and CRE-lo xP-mediated marker recycling to generate mouse embryonic stem (ES) cell lin es homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2), In this study, we describe the analysis of these ES cells,vith respect to processes known to be affected by DNA mismatch repair, ES cells homozyg ous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mecha nism is mediated by Msh2. The mutation rate of the herpes simplex virus thy midine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell line s but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells , Notably, the HSV-tk1 mutation rate was Ii-fold higher, on average, than t hat of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells, Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding r egion. Msh3-deficient cells displayed a modest (16-fold) elevation in the i nstability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 d ouble-mutant cells displayed markedly increased levels of repeat instabilit y, Targeting frequencies of nonisogenic vectors were elevated in Msh2-defic ient ES cell lines, confirming the role of Msh2 in blocking recombination b etween diverged sequences (homeologous recombination) in mammalian cells, T hese results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells.