Transgenic Mouse for DNA Damage Sensing and Signaling

用于 DNA 损伤传感和信号传导的转基因小鼠

• 批准号: 6625944
• 负责人: DAVID J CHEN
• 金额: $ 30.45万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-28 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6625944
• 关键词: CHO cells; DNA damage; DNA repair; ataxia telangiectasia; biological signal transduction; cell cycle; cell proliferation; enzyme activity; fibroblasts; gene rearrangement; gene targeting; genetic models; genetic recombination; genetically modified animals; ionizing radiation; laboratory mouse; model design /development; phosphorylation; protein kinase; protein signal sequence; radiation genetics; radiation resistance; radiation sensitivity; telomere

A cascade of molecular events is triggered in mammalian cells upon exposure to ionizing radiation (IR). These events include DNA damage recognition, damage signaling, DNA repair, and cell cycle control. Although a number of genes and gene products involved in IR responses have been identified, it is not clear how these processes are coordinated. The purpose of this proposal is to understand the inter-relationship among three major DNA damage sensing and signaling proteins: DNA-PK (DNA- dependent protein kinase), ATM (Ataxia Telangiectasia Mutated), and Nbs1 (the gene product mutated in Nijmegen breakage syndrome). We hypothesize that these DNA damage sensing and signaling proteins are coordinated in a temporal and/or spatial fashion in response to DNA damage in mammalian cells. To test this hypothesis, we plan to examine the functional relationship(s) among these proteins in primary mouse embryonic fibroblasts (MEFs) and cell lines derived from transgenic mice that are single or double knockouts (KO) for various combinations of these three proteins. We have generated in the lab or have acquired mice that are knockouts for Ku70, Ku80, DNA-PKcs, Ku80/DNA-PKcs, and ATM. To avoid embryonic and somatic lethality for Nbs1 knockout and ATM/DNA-PKcs double knockout, we plan to generate conditional knockouts for DNA-PKcs and Nbs1 using a novel one-step Tet-Off/CreLoxP strategy designed in our lab. Using these knockout and/or conditional knockout mice, we plan to elucidate 1) the role of the Ku component of DNA-PK in the modulation of ATM kinase activation by DNA damage, 2) the individual and overlapping roles of ATM and DNA-PK, and 3) the role of Nbs1 in the sensing, signaling and repair of DNA breaks. Preliminary results obtained from in vitro as well as from in vivo studies on single and double knockout mice have provided us with insights into these mechanisms. We propose to follow up these leads to obtain a clearer picture of the coordinating mechanisms between DNA-PK, ATM, and Nbs1.

当哺乳动物细胞暴露于电离辐射（IR）时，会触发一系列分子事件。 这些事件包括 DNA 损伤识别、损伤信号传导、DNA 修复和细胞周期控制。 尽管已经鉴定出许多参与IR反应的基因和基因产物，但尚不清楚这些过程是如何协调的。 本提案的目的是了解三种主要 DNA 损伤传感和信号蛋白之间的相互关系：DNA-PK（DNA 依赖性蛋白激酶）、ATM（共济失调毛细血管扩张突变）和 Nbs1（奈梅亨断裂中突变的基因产物）综合症）。 我们假设这些 DNA 损伤传感和信号蛋白以时间和/或空间方式协调，以响应哺乳动物细胞中的 DNA 损伤。 为了检验这一假设，我们计划检查原代小鼠胚胎成纤维细胞（MEF）和来自转基因小鼠的细胞系中这些蛋白质之间的功能关系，这些转基因小鼠对这三种蛋白质的各种组合进行单或双敲除（KO）。 我们在实验室中培育或获得了 Ku70、Ku80、DNA-PKcs、Ku80/DNA-PKcs 和 ATM 基因敲除的小鼠。 为了避免 Nbs1 敲除和 ATM/DNA-PKcs 双敲除造成胚胎和体细胞致死，我们计划使用我们实验室设计的一种新型一步式 Tet-Off/CreLoxP 策略来产生 DNA-PKcs 和 Nbs1 的条件敲除。使用这些敲除和/或条件敲除小鼠，我们计划阐明 1) DNA-PK 的 Ku 成分在 DNA 损伤调节 ATM 激酶激活中的作用，2) ATM 和 DNA-PK 的单独和重叠作用和 3) Nbs1 在 DNA 断裂的传感、信号传导和修复中的作用。对单敲除和双敲除小鼠进行的体外和体内研究获得的初步结果使我们对这些机制有了深入的了解。 我们建议跟踪这些线索，以更清楚地了解 DNA-PK、ATM 和 Nbs1 之间的协调机制。