RECOMBINATION AND MUTATION IN TRANSGENIC MICE

转基因小鼠的重组和突变

• 批准号: 6346151
• 负责人: JAMES STRINGER
• 金额: $ 13.47万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6346151
• 关键词: DNA damage; alkaline phosphatase; animal genetic material tag; carcinogen testing; embryonic stem cell; environmental toxicology; frameshift mutation; gene deletion mutation; gene expression; gene frequency; gene mutation; gene targeting; genetic markers; genetic promoter element; genetic recombination; genetically modified animals; histochemistry /cytochemistry; laboratory mouse; model design /development; mutagen testing; point mutation; polymerase chain reaction; reporter genes; stainings; tissue /cell culture

Environmental factors that cause genetic damage are a major public health concern. Methods to assess risk of exposure to potentially mutagenic agents have been developed, but fall short in several ways. There is a need for a method that is capable of detecting a variety of genetic changes (including homologous recombination) at the same level (individual cells) and ina the same context (intact organs) as they occur in humans. We propose to develop such a system by using a transgene that encodes an enzyme (human placental alkaline phosphatase, PAP), the activity of which can be detected in individual cells of a tissue with a histochemical stain. To allow detection of transition mutations, the PAP transgene has been altered at a single base (an A was changed to a G), which prevents production of PAP activity. The mutant PAP gene has been inserted into the genome of mice. The tissues of these animals are expected to not stain for PAP activity, except for rare cells that contain a PAP gene that has sustained a reversion mutation. Similarly, a second transgenic mouse has been made that is designed to detect frameshift mutations. This animal carries a mutant PAP gene made by insertion of a G residue into a run of G residues near the 5' end of the open reading frame encoding PAP. We propose to construct two additional types of transgenic animals designed to allow detection of homologous recombination. One such animal will detect interstitial deletion events, the other will detect mitotic recombination. These four lines of transgenic mice will be used to define the spontaneous occurrence of mutant cells in various mouse tissues. We will next determine the effects of model mutagens in these animals. Then, the transgenic animals will be crossed with other mouse strains that are cancer prone due to genetic defects, such as lack of p3, or PMS2. Through studying spontaneous and induced genetic change ina the transgenic mice, we will explore the relationship between mutational load and tumor incidence, and test the hypothesis that genetic change is more frequent in tumors and precancerous cells than in normal cells of the body.

造成遗传损害的环境因素是主要的公共卫生 忧虑。 评估暴露于潜在诱变的风险的方法 已经开发了代理商，但以多种方式跌落。 有一个 需要一种能够检测各种遗传变化的方法 （包括同源重组）在同一水平（单个细胞） 和与人类相同的上下文（完整的器官）。 我们 建议通过使用编码的转基因来开发此类系统 酶（人胎盘碱性磷酸酶，PAP），其活性 可以在具有组织化学染色的组织的单个细胞中检测到。 为了允许检测过渡突变，PAP转基因已经 在单个基础上更改（A A更改为G），这可以防止 PAP活动的产生。 突变的子基因已插入 小鼠的基因组。 这些动物的组织预计不会染色 PAP活动，除了有色包含具有PAP基因的稀有细胞 持续了一个恢复突变。 同样，第二个转基因鼠标具有 被设计为检测移码突变。 这动物 携带一个突变的子宫颈基因，将g残基插入g运行中 编码PAP的开放阅读框架5'端附近的残留物。 我们 提议构造两种旨在的转基因动物 允许检测同源重组。 这样的动物会发现 间质缺失事件，另一个将检测有丝分裂的重组。 这四行转基因小鼠将用于定义自发性 各种小鼠组织中突变细胞的发生。 我们接下来 确定模型诱变剂在这些动物中的影响。 然后， 转基因动物将与其他癌症的小鼠菌株交叉 由于缺乏P3或PMS2而易于易于遗传缺陷。 通过 研究自发性和诱导的遗传变化，是转基因小鼠，我们 将探索突变负荷与肿瘤发生率之间的关系， 并检验以下假设，即遗传变化在肿瘤中更频繁，并且 癌前细胞比体内正常细胞中的细胞。