1. The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

Correspondence to: Allan Bradley Email: abradley@sanger.ac.uk

Abstract

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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