Analysis of recombination in vivo

描述（由申请人提供）： 本提案的长期目标是开发新的工具，用于研究在小鼠发育的背景下如何通过姐妹染色单体重组调节双链断裂的修复。这项工作是可能的两个新的工具开发的细胞培养模型：第一，一种新的重组报告，允许量化特定的姐妹染色单体重组结果的流式细胞术;第二，开发的系统的严格监管的罕见切割限制性内切酶，I-SceI。该提议旨在开发携带从普遍表达的ROSA 26基因座表达的重组报告基因和从ColA 1基因座表达的I-SceI的诱导型等位基因的小鼠品系。这些模型将为研究同源重组/姐妹染色单体重组、衰老和癌症之间的关系提供强有力的新工具。我们的具体目标是：1.生成姐妹染色单体重组报告小鼠的繁殖集落。2.产生I-SceI诱导小鼠的繁殖群体。3.将这些品系杂交以产生诱导型重组报告小鼠。4.评估这些报告菌株在离体和体内姐妹染色单体重组中的性能。 公共卫生相关性： 通过称为“姐妹染色单体重组”（SCR）的过程准确修复染色体断裂对于防止染色体错误至关重要，而该过程中的缺陷会导致衰老和癌症。为了评估SCR如何在身体的不同组织中或在活体哺乳动物的发育过程中受到调节，我们开发并验证了使用流式细胞术研究姐妹染色单体重组的强大新工具。在这里，我们将使用这些相同的工具来开始研究SCR在活体小鼠中是如何调节的。

DESCRIPTION (provided by applicant): Project Summary The long-term objective of this proposal is to develop novel tools for studying how repair of double strand breaks by sister chromatid recombination is regulated in the context of mouse development. This work is made possible by two novel tools developed in cell culture models: first, a novel recombination reporter that allows quantification of specific sister chromatid recombination outcomes by flow cytometry; second, the development of a system for the tight regulation of the rare-cutting restriction endonuclease, I-SceI. This proposal aims to develop mouse strains carrying the recombination reporter expressed from the ubiquitously expressed ROSA26 locus and inducible alleles of I-SceI expressed from the ColA1 locus. These models will provide powerful new tools for studying the relationship between homologous recombination/sister chromatid recombination, aging and cancer. Our specific aims are: 1. Generate a breeding colony of sister chromatid recombination reporter mice. 2. Generate a breeding colony of I-SceI-inducible mice. 3. Cross these strains to generate inducible recombination reporter mice. 4. Assess the performance of these reporter strains in sister chromatid recombination both ex vivo and in vivo. PUBLIC HEALTH RELEVANCE: Project Narrative The accurate repair of chromosome breaks by a process termed "sister chromatid recombination" (SCR) is critical for preventing chromosome errors, and defects in this process contribute to aging and cancer. To assess how SCR is regulated in different tissues of the body, or during the development of a living mammal, we have developed and validated powerful new tools for studying sister chromatid recombination using flow cytometry. Here, we will use these same tools to begin to study how SCR is regulated in a live mouse.