RECOMBINATION AND MUTATION IN TRANSGENIC MICE

转基因小鼠的重组和突变

• 批准号: 6106287
• 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: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6106287
• 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.

造成遗传损伤的环境因素是重大公共卫生问题 忧虑。 评估潜在致突变暴露风险的方法 代理已经被开发出来，但在几个方面还存在不足。 有一个 需要一种能够检测多种遗传变化的方法 （包括同源重组）同一水平（单个细胞） 并且在与人类发生相同的环境（完整的器官）中。 我们 建议通过使用编码的转基因来开发这样的系统 酶（人胎盘碱性磷酸酶，PAP），其活性 可以通过组织化学染色在组织的单个细胞中检测到。 为了检测过渡突变，PAP 转基因已被 在单个碱基处发生改变（A 更改为 G），这可以防止 PAP 活动的产生。 突变的 PAP 基因已被插入 小鼠的基因组。 这些动物的组织预计不会被染色 PAP 活性，但含有 PAP 基因的稀有细胞除外 持续回复突变。 同样，第二只转基因小鼠也有 旨在检测移码突变。 这种动物 携带突变的 PAP 基因，该基因是通过将 G 残基插入到 G 序列中而制成的 编码 PAP 的开放阅读框 5' 端附近的残基。 我们 提议构建另外两种类型的转基因动物，旨在 允许检测同源重组。 一只这样的动物会检测到 间质缺失事件，另外会检测有丝分裂重组。 这四种转基因小鼠品系将用于定义自发性 小鼠各种组织中出现突变细胞。 我们接下来将 确定模型诱变剂对这些动物的影响。 然后， 转基因动物将与其他癌症小鼠品系杂交 容易因遗传缺陷而导致，例如缺乏 p3 或 PMS2。 通过 研究转基因小鼠自发和诱导的遗传变化，我们 将探索突变负荷与肿瘤发生率之间的关系， 并检验基因变化在肿瘤中更频繁的假设 癌前细胞多于体内正常细胞。