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Molecular and Cellular Biology, January 2000, p. 149-157, Vol. 20, No. 1
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Genetic Analysis of Mouse Embryonic Stem Cells Bearing Msh3 and Msh2 Single and Compound Mutations

Alejandro Abuin,1,dagger HeJu Zhang,1 and Allan Bradley1,2,*

Department of Molecular and Human Genetics1 and Howard Hughes Medical Institute,2 Baylor College of Medicine, Houston, Texas 77030

Received 20 April 1999/Returned for modification 19 June 1999/Accepted 10 September 1999

We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines 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 with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that 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 region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells.

* Corresponding author. Mailing address: Department of Molecular and Human Genetics, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-6671. Fax: (713) 798-8142. E-mail: abradley{at}bcm.tmc.edu.

dagger Present address: Lexicon Genetics Inc., The Woodlands, TX 77381.

Molecular and Cellular Biology, January 2000, p. 149-157, Vol. 20, No. 1
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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