ENVIRONMENTAL TOXICOLOGY USING TRANSGENIC MOUSE MODELS

使用转基因小鼠模型进行环境毒理学

• 批准号: 6382104
• 负责人: GLEN K ANDREWS
• 金额: $ 29.85万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-05 至 2002-08-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6382104
• 关键词: cadmium; environmental toxicology; enzyme mechanism; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic promoter element; genetic regulatory element; genetically modified animals; histopathology; immunocytochemistry; ion exchange chromatography; laboratory mouse; metal poisoning; metallothionein; microinjections; model design /development; northern blottings; nucleic acid hybridization; oxidative stress; polymerase chain reaction; protein biosynthesis; protein structure function; solution hybridization; toxicant screening; transcription factor; western blottings

DESCRIPTION (Adapted from the APPLICANT'S ABSTRACT): The overall long-term objective of these studies is to develop and use transgenic animal model systems to study environmental health-related issues. Specifically, our studies focus on the metallothioneins (MTs) and transcription factors that regulate MT genes. We propose to determine the molecular mechanisms regulating expression of MT genes in response to cadmium (Cd) and oxidative stresses, and the functional significance of MT in protection from oxidative stress. Cd is a widespread environmental pollutant and many xenobiotics cause damaging oxidative stress. MTs represent the best documented intracellular heavy metal (Zn, Cu, Cd) binding proteins, but they can also scavenge hydroxyl free radicals, and dismutate superoxide anions. Transcriptional induction of the mouse MT-I gene by oxidative stress involves activation of MTF-1 binding to metal responsive elements (MRE) in the promoter, as well as a composite USF/antioxidant response promoter element (USF/ARE). Preliminary evidence indicates that Cd induction is also mediated, in part, by this element whereas Zn induction is not. Little is known about the structure and function of MTF-1 or the USF/ARE. Transgenic mice that over-express MT or that have targeted ablations of MT-I/MT-II gene function have been created and will be used to examine the roles of these proteins in protection from oxidative stress. The specific aims of this proposal are to: 1) Determine the molecular mechanisms by which oxidative stresses and metals activate MTF-1; 2) Explore the functional roles of the USF/antioxidant response element in the mouse MT-I promoter during induction by oxidative stress and Cd; 3) Examine protein interactions with the USF/ARE during oxidative stress and Cd induction of gene expression; and 4) Analyze resistance to oxidative stress in transgenic mice that over-express MT or that have targeted ablations of the MT genes. Molecular (mutagenesis, EMSA, footprinting, transfection) and biochemical (Zn titration analysis, immunoprecipitation, binding site chromatography) approaches will be used to define structure-function relationships for MTF-1 (Zn binding, DNA binding, protein interactions and transactivation). Structure-function of the USF/ARE will be defined by mutagenesis, transfection assays, and protein binding assays. Resistance to oxidative stress in transgenic mice will be monitored by histopathology and serum enzyme levels.

描述（根据申请人的摘要改编）：总体长期 这些研究的目的是开发和使用转基因动物模型 研究与环境健康相关问题的系统。 具体来说，我们的 研究重点是金属硫硫蛋白（MTS）和转录因子 调节MT基因。 我们建议确定分子机制 调节MT基因对镉（CD）的表达和氧化 应力，以及MT在保护免受氧化方面的功能意义 压力。 CD是一种广泛的环境污染物和许多异生物污染物 导致破坏性氧化应激。 MTS代表了最好的记录 细胞内重金属（Zn，Cu，cd）结合蛋白，但也可以 清除羟基自由基，并拆除超氧化物阴离子。 通过氧化应激对小鼠MT-I基因的转录诱导 涉及激活MTF-1与金属响应元件（MRE）的激活 启动子以及复合USF/抗氧化剂反应启动子 元素（USF/AR）。 初步证据表明CD诱导也是 部分是由该元素介导的，而锌诱导不是。 几乎没有 知道MTF-1或USF/的结构和功能。 转基因 过表达的MT或已针对MT-I/MT-II基因的消融的小鼠 功能已被创建，将用于检查这些功能 蛋白质免受氧化应激的保护。 这个特定的目的 建议是：1）确定氧化的分子机制 应力和金属激活MTF-1； 2）探索功能作用 诱导过程中小鼠MT-1启动子中的USF/抗氧化剂响应元件 通过氧化应激和CD； 3）检查与USF/IS的蛋白质相互作用 在基因表达的氧化应激和CD诱导过程中； 4）分析 过表达MT或 针对MT基因的消融。 分子（诱变，EMSA，，， 足迹，转染）和生化（锌滴定分析， 免疫沉淀，结合位点色谱法）方法将用于 定义MTF-1的结构 - 功能关系（Zn结合，DNA结合， 蛋白质相互作用和反式激活）。 结构功能 USF/将由诱变，转染测定和蛋白质定义 绑定测定。 转基因小鼠中对氧化应激的抗性将是 通过组织病理学和血清酶水平监测。