Chen Y, Yee D, Dains K, Chatterjee A, Cavalcoli J, Schneider E, Om J, Woychik R P, Magnuson T
Department of Genetics, Case Western Reserve University, Cleveland, Ohio, USA.
Nat Genet. 2000 Mar;24(3):314-7. doi: 10.1038/73557.
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. In fact, several genome-wide and region-specific 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.
产生突变的能力是进行功能基因分析的前提条件。尽管长期以来一直将小鼠用作基因分析的模型系统，但科学界尚未针对大多数小鼠基因生成多个等位基因的全面集合。小鼠常用的化学诱变剂是N-乙基-N-亚硝基脲（ENU），这是一种烷化剂，主要导致DNA中的碱基替换，因此能够获得功能完全丧失、部分丧失以及功能获得的等位基因。旨在检测隐性突变的特定基因座测试表明，ENU是小鼠中最有效的诱变剂，突变率约为每1000个配子中有1个发生突变。事实上，已经基于表型进行了多项全基因组和区域特异性筛选。然而，随着人类和小鼠基因组计划即将完成，现在更强调基因型驱动的遗传学——从序列到突变体。为了利用ENU的诱变性及其产生一系列等位基因突变的能力，我们开发了一种利用小鼠胚胎干细胞（ES细胞）产生突变的互补方法。我们表明，可以实现高突变频率，并且调节DNA修复活性可以提高这一频率。经处理的细胞保留种系能力，从而使该方法适用于高效生成一系列等位基因突变，这些突变对于精细剖析生物途径至关重要。
