Allelic Series of Genomic Modifications in ES Cells

描述(申请人提供)：产生突变的能力是 功能遗传分析的先决条件。尽管有很长的使用历史 以小鼠为模型系统进行遗传分析，全面收集 因为多数基因的多重等位基因是不存在的。的化学诱变剂 小鼠的选择是N-乙基-N-亚硝脲(ENU)，这是一种烷基化试剂，可以 主要导致DNA中的碱基替换，因此允许恢复 完全和部分丢失，以及功能等位基因的获得。特定基因座 旨在检测隐性突变的测试表明，ENU是最多的 一种高效的小鼠诱变剂，其突变率约为1个突变 筛选出200~1000个配子。事实上，有几个基因组范围和区域特定的 基于表型的筛选已经成功地进行了。然而， 人类和小鼠基因组计划即将完成 强调由基因决定的遗传学--从序列到突变 理解功能。利用ENU及其基因的致突变性 创造等位基因突变系列的能力，一个互补的方法是 被描述为使用小鼠胚胎干细胞产生突变。一个 可以实现高突变频率，并调节DNA修复活动 可以提高这一频率。处理后的细胞保留了生殖系能力， 从而使该方法适用于高效、高吞吐量 产生对微调解剖至关重要的等位基因突变系列 生物途径的。这项提案中概述的实验将是下一步 通过应用这一平台，迈向该方法的全基因组应用 等位基因表型的生产和分析技术 ENU诱导的Smad2和Smad4基因座的一系列突变。此外，a 建议进行一系列实验，以将该技术推广到整个动物 使用已经在TGFbeta中妥协的ES细胞的基于表型的筛查 信号通路。能够对频率和类型进行调制和监控 突变的产生，再加上突变检测的进步，使得 在ES细胞中进行突变是对现有策略的有力补充 提供一个扩展的小鼠突变剧目。

DESCRIPTION (provided by applicant): The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using the mouse as a model system for genetic analysis, a comprehensive collection for multiple alleles of most genes does not exist. The chemical mutagen of choice for the 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 mutation per 200 to 1000 gametes screened. In fact, several genome-wide and region-specific phenotype-based screens have been carried out with success. However, the anticipation of the completion of the human and mouse genome projects now emphasizes genotype-driven genetics--from sequence to mutations for understanding function. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, a complementary approach is described for generating mutations using mouse embryonic stem (ES) cells. A high mutation frequency can be achieved, and modulating DNA repair activities can enhance this frequency. The treated cells retain germ line competency, thereby rendering this approach applicable for efficient, high-throughput generation of allelic series of mutations pivotal for a fine-tuned dissection of biological pathways. The experiments outlined in this proposal take the next step toward genome wide application of this approach by applying this platform technology to production and analysis of phenotypes resulting from an allelic series of ENU-induced mutations in the Smad2 and Smad4 loci. In addition, a series of experiments are proposed to extend the technology toward whole animal phenotype-based screens using ES cells already compromised in the Tgfbeta signaling pathway. The ability to modulate and monitor the frequency and type of mutation produced, coupled with advancements in mutation detection, makes mutagenesis in ES cells a powerful addition to the existing strategies to deliver an expanded repertoire of mouse mutants.