Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene

Kosuke Yusa, Kyoji Horie, Gen Kondoh, Michiyoshi Kouno, Yusuke Maeda, Taroh Kinoshita and Junji Takeda ()
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Kosuke Yusa: Osaka University
Kyoji Horie: Osaka University
Gen Kondoh: Osaka University
Michiyoshi Kouno: Osaka University
Yusuke Maeda: Osaka University
Taroh Kinoshita: Osaka University
Junji Takeda: Osaka University

Nature, 2004, vol. 429, issue 6994, 896-899

Abstract: Abstract The chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. Cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity1,2,3. Here we have used a tetracycline-regulated Blm allele (Blmtet) to introduce bi-allelic mutations across the genome in mouse embryonic stem (ES) cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. We considered that the phenotype of ES cells bearing bi-allelic mutations would be maintained after withdrawal of the tetracycline analogue doxycycline. Indeed, a combination of N-ethyl-N-nitrosourea mutagenesis and transient loss of Blm expression enabled us to generate an ES cell library with genome-wide bi-allelic mutations. The library was evaluated by screening for mutants of glycosylphosphatidylinositol-anchor biosynthesis, which involves at least 23 genes distributed throughout the genome. Mutants derived from 12 different genes were obtained and two unknown mutants were simultaneously isolated. Our results indicate that phenotype-based genetic screening with Blmtet is very efficient and raises possibilities for identifying gene functions in ES cells.

Date: 2004
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DOI: 10.1038/nature02646

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