Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene
Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene
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DOI：
10.1038/nature02646
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发表时间：
2004-06-24
期刊：
影响因子：
64.8
通讯作者：
Takeda, J
中科院分区：
文献类型：
--
作者：
Yusa, K;Horie, K;Takeda, J
在哺乳动物系统中基于表型的遗传筛选的主要限制是基因组的二倍体性质。Bloom综合征基因（Blm）缺陷的细胞显示出增加的杂合性丢失率（1-3）。在这里，我们已经使用四环素调节的Blm等位基因（Blm（泰特））在小鼠胚胎干（ES）细胞的基因组中引入双等位基因突变。Blm表达的瞬时缺失不仅诱导姐妹染色单体之间的同源重组，而且诱导同源染色体之间的同源重组。我们认为，ES细胞的表型轴承双等位基因突变后，四环素类似物多西环素撤出将保持。事实上，N-乙基-N-亚硝基脲诱变和Blm表达的瞬时丧失的组合使我们能够产生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锚生物合成的突变体来评估文库，所述突变体涉及分布在整个基因组中的至少23个基因。获得了12个不同基因的突变体，同时分离到两个未知突变体。我们的研究结果表明，基于表型的遗传筛选与Blm（泰特）是非常有效的，并提出了可能性，在ES细胞中识别基因功能。
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.