ENVIRONMENTAL TOXICOLOGY USING TRANSGENIC MOUSE MODELS

这些研究的总体长期目标是发展 转基因小鼠的染色可以作为了解 环境污染物毒理学。具体地说，这些 研究将集中在金属硫蛋白(MTS)上，并将 演示；a)调节这些基因表达的机制 对具有毒性的重要化学品的反应，以及b) 这些蛋白质的功能。MT是小的，富含半胱氨酸，很重 金属结合蛋白。它们代表了记录最好的 细胞内重金属(锌、铜、镉)结合蛋白， 并被认为在保护免受金属伤害方面起着关键作用 毒性。此外，MTS可能在保护 烷化剂、化疗药物和辐射。MTS 可以清除自由基，因此可以提供保护，防止 引起氧化应激的有毒化学物质。大多数，如果不是全部的话， 上述化学物质也可增强MT的表达 基因。然而，除了金属离子的反应外，我们知之甚少。 关于MT基因发生诱导的机制。 尽管体外研究表明MTS是重要的成分 细胞对各种毒素的反应，我们的能力 准确地定义它们在体内的功能角色受到一种 缺乏表现出缺陷(突变)影响的小鼠品系 MT基因或蛋白质结构-功能。这样做的具体目的是 因此，建议建立转基因小鼠品系： 1)为了确定诱导剂，如氧化应激 诱导剂，增强小鼠MT基因的表达，2) 显示结构性增高或改变的组织特异性 MT基因的表达，以及3)靶向消融 MT基因。顺式作用启动子序列将在 受精卵显微注射获得转基因小鼠品系 与火蝇连锁的MT启动子缺失突变体的小鼠卵 荧光素酶报告基因。转基因小鼠构成了 在许多组织中过度表达MT基因将使用 病毒增强子驱动的MT表达，以及转基因小鼠 以组织特异性的方式结构性地过度表达MT基因 将使用白蛋白(肝脏特异性)增强子驱动创建 MT的表达。鸡MT基因将用于研究 涉及到过表达是因为鸡MT的mRNA和蛋白 很容易与小鼠MT的mRNAs和蛋白质区分开来。 MT基因缺失或消除的胚胎干细胞 将通过靶向整合转基因来创建表达 将DNA导入MT基因座。靶向消融MT-1和/或 将执行MT-11基因。稳定的转基因品系将是 从显微注射这些基因产生的生殖系嵌合体中获得 胚胎干细胞进入囊胚期。基因表达将由以下因素决定 用Northern印迹、溶液杂交、 和逆转录酶聚合酶链式反应，并通过 蛋白质合成与积累的分析 脉冲标记、免疫印迹、免疫细胞化学和阴离子 交换层析。转基因菌株将被分析 改变了对几种药物的肝毒性作用的抵抗力， 包括镉和氧化应激诱导剂，通过 组织病理学和血清酶的定量。

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.