Allelic Series of Genomic Modifications in ES Cells

ES 细胞中基因组修饰的等位基因系列

• 批准号: 6696933
• 负责人: TERRY MAGNUSON
• 金额: $ 32.74万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-21 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6696933
• 关键词: alleles; biological signal transduction; biotechnology; cryopreservation; developmental genetics; embryonic stem cell; functional /structural genomics; gene expression; genetic library; genetic mapping; genetic techniques; high performance liquid chromatography; high throughput technology; microarray technology; phenotype; point mutation; transfection

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.

描述（由申请人提供）：产生突变的能力是 功能性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 大多数基因的多重等位基因是不存在的。化学诱变剂 小鼠的选择是N-乙基-N-亚硝基脲（ENU），这是一种烷基化剂， 主要导致DNA中的碱基替换，因此，允许恢复 功能等位基因的完全和部分缺失以及获得。特定基因座 设计检测隐性突变的试验表明，ENU是最 在小鼠中的有效诱变剂，突变率约为1个突变/ 筛选200至1000个配子。事实上，几个基因组范围和区域特异性 基于表型的筛选已经成功地进行。但 人类和老鼠基因组计划的完成， 强调基因型驱动的遗传学--从序列到突变， 理解功能。利用ENU的致突变性及其 创造等位基因系列突变的能力，互补的方法是 描述了使用小鼠胚胎干（ES）细胞产生突变。一 可以实现高突变频率，并调节DNA修复活性 可以提高这个频率。经处理的细胞保留了生殖系能力， 从而使该方法适用于高效、高通量的 产生对微调解剖至关重要的等位基因系列突变 的生物学途径。本提案中概述的实验采用了下一个 通过应用该平台，朝着该方法基因组广泛应用迈出了一步 产生和分析由等位基因产生的表型的技术 一系列ENU诱导的Smad 2和Smad 4基因座突变。另外还有按 提出了一系列实验，以将该技术扩展到整个动物 基于表型的筛选，使用已经在Tgf β中受损的ES细胞 信号通路调制和监控频率和类型的能力 突变的产生，再加上突变检测的进步， ES细胞中的诱变是对现有策略的有力补充， 提供了一个扩大的小鼠突变体库。