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Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells


Phenotype-driven recessive genetic screens in diploid organisms require a strategy to render the mutation homozygous. Although homozygous mutant mice can be generated by breeding, a reliable method to make homozygous mutations in cultured cells has not been available, limiting recessive screens in culture. Cultured embryonic stem (ES) cells1 provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Here we have exploited the high rate of mitotic recombination in Bloom's syndrome protein (Blm)-deficient ES2 cells to generate a genome-wide library of homozygous mutant cells from heterozygous mutations induced with a revertible gene trap3 retrovirus. We have screened this library for cells with defects in DNA mismatch repair (MMR), a system that detects and repairs base–base mismatches4. We demonstrate the recovery of cells with homozygous mutations in known and novel MMR genes. We identified Dnmt1(ref. 5) as a novel MMR gene and confirmed that Dnmt1-deficient ES cells exhibit micro-satellite instability6, providing a mechanistic explanation for the role of Dnmt1 in cancer. The combination of insertional mutagenesis in Blm-deficient ES cells establishes a new approach for phenotype-based recessive genetic screens in ES cells.

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Figure 1: Mechanisms and components used in the screen.
Figure 2: Molecular analysis of Msh6 gene trap mutants.
Figure 3: Analysis of Dnmt1 gene trap mutants.
Figure 4: Slippage assay for micro-satellite instability.

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We would like to thank G. Luo and X. Wang for discussions at an early phase in the design of this screen, F. Law and A. Beasley for help with tissue culture and B. Skarnes, P. Liu, D. Adams and J. Jonkers for their comments on this manuscript. This work was supported by the Wellcome Trust.

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Correspondence to Allan Bradley.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Table

This table lists gene trap ES cell clones that are 6TG resistant, but the phenotype cannot be reverted by Cre-mediated recombination. (PDF 57 kb)

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Guo, G., Wang, W. & Bradley, A. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells. Nature 429, 891–895 (2004).

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