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. 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

Kyoji Horie  1 , Chikara Kokubu, Junko Yoshida, Keiko Akagi, Ayako Isotani, Akiko Oshitani, Kosuke Yusa, Ryuji Ikeda, Yue Huang, Allan Bradley, Junji Takeda
Affiliations

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

Kyoji Horie et al. Nat Methods. .

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.

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