A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening
A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening
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DOI：
10.1038/nmeth.1739
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发表时间：
2011-12-01
期刊：
影响因子：
48
通讯作者：
Takeda, Junji
中科院分区：
文献类型：
--
作者：
Horie, Kyoji;Kokubu, Chikara;Takeda, Junji
小鼠胚胎干细胞（ESCs）的全基因组突变是一种强大的工具，但哺乳动物基因组的二倍体特性阻碍了其在隐性遗传筛选中的应用。我们以前报道过一种方法，通过四环素依赖的Bloom综合征基因的短暂破坏，从杂合突变体诱导纯合突变体ESCs。然而，我们无法从大量杂合突变细胞中纯化纯合突变体，限制了其应用。在这里，我们开发了一种快速富集纯合突变小鼠ESCs的策略，并证明了其用于基于细胞的表型分析的可行性。方法采用g418 +嘌呤霉素双选择富集纯合子，单核苷酸多态性分析鉴定纯合子。我们将这种简单的方法与基因诱变相结合，构建了一个包含138个突变系的纯合子突变体ESC库，并证明了其在表型驱动遗传筛选中的应用。
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.