Yusa Kosuke, Horie Kyoji, Kondoh Gen, Kouno Michiyoshi, Maeda Yusuke, Kinoshita Taroh, Takeda Junji
Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
Nature. 2004 Jun 24;429(6994):896-9. doi: 10.1038/nature02646.
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. 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.
在哺乳动物系统中，基于表型的基因筛选的主要限制是基因组的二倍体性质。布卢姆氏综合征基因（Blm）缺陷的细胞显示杂合性丧失率增加。在这里，我们使用了四环素调控的Blm等位基因（Blm(tet)）在小鼠胚胎干细胞（ES细胞）中引入全基因组的双等位基因突变。短暂丧失Blm表达不仅会诱导姐妹染色单体之间的同源重组，还会诱导同源染色体之间的同源重组。我们认为，携带双等位基因突变的ES细胞的表型在撤除四环素类似物强力霉素后会得以维持。事实上，N-乙基-N-亚硝基脲诱变与Blm表达的短暂丧失相结合，使我们能够生成一个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对该文库进行了评估，糖基磷脂酰肌醇锚生物合成涉及至少23个分布在整个基因组中的基因。获得了来自12个不同基因的突变体，并同时分离出两个未知突变体。我们的结果表明，用Blm(tet)进行基于表型的基因筛选非常有效，并为鉴定ES细胞中的基因功能带来了可能性。
