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Nat Methods. 2011 Oct 23;8(12):1071-7. doi: 10.1038/nmeth.1739.

A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening.

Author information

  • 1Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan. horie@mr-envi.med.osaka-u.ac.jp

Abstract

Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.

PMID:
22020066
[PubMed - indexed for MEDLINE]
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