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首页> 外文期刊>Nature >Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene
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Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene

机译：通过调节布鲁姆氏综合征基因的破坏，在ES细胞中进行全基因组表型分析

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摘要

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 heterozygosity(1-3). Here we have used a tetracycline-regulated Blm allele (Blm(tet)) 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 Blm(tet) is very efficient and raises possibilities for identifying gene functions in ES cells.
机译：在哺乳动物系统中基于表型的遗传筛选的主要局限性是基因组的二倍体性质。缺乏布卢姆综合症基因（Blm）的细胞显示杂合性丧失率增加（1-3）。在这里，我们已经使用四环素调节的Blm等位基因（Blm（tet））在小鼠胚胎干（ES）细胞中通过基因组引入双等位基因突变。 Blm表达的瞬时丧失不仅在姐妹染色单体之间诱导同源重组，而且在同源染色体之间诱导同源重组。我们认为，在退出四环素类似物多西环素后，具有双等位基因突变的ES细胞表型将得以维持。确实，N-乙基-N-亚硝基脲诱变和Blm表达的瞬时丧失相结合，使我们能够生成具有全基因组双等位基因突变的ES细胞文库。通过筛选糖基磷脂酰肌醇固定生物的突变体来评估该文库，该突变体涉及分布在整个基因组中的至少23个基因。获得了来自12个不同基因的突变体，并同时分离了两个未知突变体。我们的结果表明，使用Blm（tet）进行基于表型的遗传筛选非常有效，并为鉴定ES细胞中的基因功能提供了可能性。

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