Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells
Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells
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DOI：
10.1038/73557
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发表时间：
2000-03-01
期刊：
影响因子：
30.8
通讯作者：
Magnuson, T
中科院分区：
文献类型：
--
作者：
Chen, YJ;Yee, D;Magnuson, T
产生突变的能力是功能性遗传分析的先决条件。尽管使用小鼠作为遗传分析模型系统的历史很长，但科学界还没有为大多数小鼠基因生成多个等位基因的全面集合。小鼠选择的化学诱变剂是n -乙基-n -亚硝基脲（ENU）。一种烷基化剂，主要引起DNA中的碱基替换，因此允许恢复完全和部分失去功能的等位基因，以及获得功能的等位基因。设计用于检测隐性突变的特异性位点测试表明，ENU是小鼠中最有效的诱变原，突变率约为1 / 1000配子（2,3）。事实上，已经开展了几种基于表型的全基因组（4-7）和区域特异性（8-10）筛选。然而，对人类和小鼠基因组计划完成的预期，现在强调基因型驱动的遗传学——从序列到突变体。为了利用ENU的致突变性及其产生等位基因系列突变的能力，我们开发了一种利用小鼠胚胎干细胞（ES）产生突变的补充方法。我们发现可以实现高突变频率，并且调节dna修复活动可以提高这种频率。处理后的细胞保留了种系能力，因此使这种方法适用于有效产生等位基因系列突变，这对生物途径的微调解剖至关重要。
The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU). an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles', Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes(2,3). In fact, several genome-wide(4-7) and region-specific(8-10) screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics-from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.