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Title: Recessive genetic screen for mismatch repair components in Blm-deficient ES cells
Author: Guo, G.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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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) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell, as well as genes involved in differentiation. It has been established that in Blm-deficient cells, homozygous daughter cells can be readily segregated from cells carrying heterozygous mutations, presumably through mitotic recombination between non-sister chromatids. In this study, I have exploited the high rate of mitotic recombination in Blm-deficient ES cells to generate a genome wide library of homozygous mutant cells from heterozygous mutations induced with a revertible gene trap retrovirus. This library is composed of nearly 10,000 individual gene trap clones. To further investigate the use of this library, a recessive genetic screen has been carried out to identify cells with defects in DNA mismatch repair (MMR) that exhibit resistance to 6-thioguanine. Multiple homozygous mutants in mismatch repair homologue 6 (Msh6) were recovered, providing confirmation of the effectiveness of this recessive genetic screen. Dnmt1 was recovered as a novel MMR gene from this screen. It was verified that Dnmt1-deficient ES cells exhibit micro-satellite instability. Dnmt1 mutant mice are predisposed to certain type of cancers. The finding that Dnmt1 is a novel MMR gene provides new insights into the mechanistic role of Dnmt1 in cancer. Importantly, the combination of insertional mutagenesis in Blm-deficient ES cells opens a new approach for phenotype based recessive genetic screens in ES cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available