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: 2154517
• 负责人: GLEN K ANDREWS
• 金额: $ 25.9万
• 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 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2154517
• 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

The overall long-term objective of these studies is to develop transgenic stains of mice to serve as tools for understanding the toxicology of environmental pollutants. Specifically, these studies will focus on the metallothioneins (MTs), and will demonstrate; a) mechanisms regulating expression of these genes in response to toxicologically important chemicals, and b) the functions of these proteins. The MTs are small cysteine-rich heavy metal binding proteins. They represent the best documented intracellular heavy metal (zinc, copper, cadmium) binding proteins, and are considered to play a pivotal role in protection from metal toxicity. In addition, MTs may play a role in protection from alkylating agents, chemotherapeutic agents, and irradiation. MTs can scavenge free radicals, and may thus provide protection from toxic chemicals that induce oxidative stress. Most, if not all, of the aforementioned chemicals also enhance expression of the MT genes. However, except for the metal ion response, little is known about the mechanisms by which induction of the MT genes occurs. Although in vitro studies suggest that MTs are important components of the cell's responses to a variety of toxins, our ability to precisely define their functional roles in vivo is hampered by a lack of strains of mice that exhibit defects (mutations) effecting MT gene or protein structure-function. The specific aims of this proposal are, therefore, to establish strains of transgenic mice: 1) in order to determine how inducers, such as oxidative stress inducing agents, enhance expression of the mouse MT genes, 2) that display constitutively heightened or altered tissue-specific expression of the MT genes, and 3) that have targeted ablations of the MT genes. Cis-acting promoter sequences will be mapped in transgenic mouse strains created by microinjection into fertilized mouse eggs of MT promoter deletion mutants linked to a fire fly luciferase reporter gene. Transgenic mice that constitutively over-express the MT gene in many tissues will be created using viral enhancer-driven expression of MT, and transgenic mice that constitutively over-express the MT gene in a tissue-specific manner will be created using albumin (liver specific) enhancer-driven expression of MT. The chicken MT gene will be utilized in studies involving overexpression because the chicken MT mRNA and protein are readily distinguishable from the mouse MT mRNAs and proteins. Embryonic stem (ES) cells with reduced or eliminated MT gene expression will be created by targeted integration of transfected DNA into the MT gene locus. Targeted ablation of the MT-1 and/or MT-11 genes will be performed. Stable transgenic lines will be obtained from germ line chimeras created by microinjection of these ES cells into blastocysts. Gene expression will be determined by analysis of RNA using Northern blotting, solution hybridization, and the reverse transcriptase-polymerase chain reaction, and by analysis of protein synthesis and accumulation using pulse-labeling, western blotting, immunocytochemistry and anion exchange chromatography. Transgenic strains will be analyzed for altered resistance to the hepatotoxic effects of several agents, including cadmium and inducers of oxidative stress, by histopathology and quantitation of serum enzymes.

这些研究的总体长期目标是发展 小鼠的转基因污渍，作为理解的工具 环境污染物的毒理学。 具体来说，这些 研究将重点放在金属硫硫蛋白（MTS）上，并将 证明; a）调节这些基因表达的机制 对毒理学重要化学物质的反应，b） 这些蛋白质的功能。 MTS是小型半胱氨酸的重量 金属结合蛋白。 它们代表了最好的记录 细胞内重金属（锌，铜，镉）结合蛋白， 并被认为在保护金属中起着关键作用 毒性。 此外，MTS可能在保护中发挥作用 烷基化剂，化学治疗剂和辐照。 MTS 可以清除自由基，因此可以提供保护 诱导氧化应激的有毒化学物质。 大多数（如果不是全部）， 上述化学物质也增强了MT的表达 基因。 但是，除了金属离子响应以外，知之甚少 关于MT基因诱导的机制。 尽管体外研究表明MT是重要的组成部分 细胞对各种毒素的反应，我们的能力 精确定义其在体内的功能作用受到A的阻碍 缺乏表现出缺陷（突变）的小鼠菌株 MT基因或蛋白质结构功能。 这个特定的目的 因此，提案是建立转基因小鼠菌株的建议： 1）为了确定诱导剂（例如氧化应激）的诱导剂 诱导剂，增强小鼠MT基因的表达，2） 显示出组成性增强或改变的组织特异性 MT基因的表达和3）针对消融的表达 MT基因。 顺式作用启动子序列将在 通过显微注射施肥而产生的转基因小鼠菌株 MT启动子缺失突变体的鼠标卵与火蝇有关 荧光素酶报告基因。 组成性的转基因小鼠 过表达许多组织中的MT基因 病毒增强子驱动的MT和转基因小鼠的表达 以组织特异性方式过表达MT基因 将使用白蛋白（肝特异性）增强子驱动创建 Mt的表达。 鸡MT基因将用于研究 涉及过表达，因为鸡肉mt mRNA和蛋白质 很容易与小鼠mt mRNA和蛋白质区分开。 胚胎茎（ES）细胞，降低或消除了MT基因 表达将通过针对转染的目标集成创建 DNA进入MT基因基因座。 目标消融MT-1和/或 将执行MT-11基因。 稳定的转基因线将是 从通过对这些的微注射产生的种系嵌合体获得 ES细胞成胚泡。 基因表达将由 使用北印迹，溶液杂交分析RNA， 以及逆转录酶 - 聚合酶链反应，以及 使用蛋白质合成和积累分析 脉搏标记，蛋白质印迹，免疫细胞化学和阴离子 交换色谱法。 转基因菌株将被分析 改变了几种药物的肝毒性作用的抗性， 包括镉和氧化应激诱导剂， 血清酶的组织病理学和定量。