ENVIRONMENTAL TOXICOLOGY USING TRANSGENIC MOUSE MODELS

这些研究的总体长期目标是发展 转基因小鼠染色作为工具，了解 环境污染物毒理学。 具体来说，这些 研究将集中在金属硫蛋白（MT），并将 证明; a）调节这些基因表达的机制 对毒理学上重要的化学品的反应，和B） 这些蛋白质的功能。 MT是小的富含半胱氨酸的重链 金属结合蛋白 他们代表了最好的记录 细胞内重金属（锌、铜、镉）结合蛋白， 并且被认为在金属防护方面起着关键作用 毒性 此外，多边贸易体制还可发挥保护作用， 烷化剂、化疗剂和辐射。 MTS 可以抑制自由基，因此可以提供保护， 有毒化学物质会导致氧化应激。 大部分，如果不是全部， 上述化学物质也能增强MT的表达， 基因. 然而，除了金属离子的响应，知之甚少 关于MT基因诱导发生的机制。 尽管体外研究表明MT是 细胞对各种毒素的反应，我们的能力， 精确定义它们在体内的功能作用受到阻碍， 缺乏表现出缺陷（突变）的小鼠品系， MT基因或蛋白质结构-功能。 具体目标是 因此，建议建立转基因小鼠品系： 1)为了确定诱导物，如氧化应激， 诱导剂，增强小鼠MT基因的表达，2） 显示组成性增强或改变的组织特异性 MT基因的表达，和3）具有靶向消融的MT基因， MT基因 顺式作用启动子序列将在 通过显微注射到受精卵中产生的转基因小鼠品系 与火蝇相连的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， 在许多组织中过表达MT基因将被创建， 病毒增强子驱动的MT表达，以及 以组织特异性方式组成型过表达MT基因 将使用白蛋白（肝脏特异性）增强子驱动 表达MT。 鸡MT基因将用于研究 因为鸡的MT mRNA和蛋白质 容易与小鼠MT mRNA和蛋白质区分。 MT基因减少或消除的胚胎干细胞 将通过转染的细胞的靶向整合来产生表达。 DNA插入MT基因位点。 MT-1靶向消融和/或 将进行MT-11基因检测。 稳定的转基因株系将 从生殖系嵌合体获得，所述生殖系嵌合体通过显微注射这些 胚胎干细胞进入胚泡。 基因表达将由 使用北方印迹，溶液杂交， 和逆转录酶-聚合酶链反应， 蛋白质合成和积累的分析， 脉冲标记、蛋白质印迹、免疫细胞化学和阴离子 交换层析 将分析转基因菌株的 对几种药物的肝毒性作用的抗性改变， 包括镉和氧化应激诱导剂， 组织病理学和血清酶定量。

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.