LIVER SPECIFIC, INDUCIBLE TRANSGENE AND HEPATOTOXICITY

描述：许多外源性毒素改变了一个亚类的表达， 通过首先诱导基因的表达， 编码转录因子ATF 3，其是bZIP（基本的 区域-亮氨酸拉链）蛋白和ATF/CREB家族的成员， 转录因子 用于ATF 3结合的共有序列已经被克隆。 在许多肝脏特异性基因启动子中鉴定，特别是那些 被认为调节鸟氨酸转氨甲酰酶，醛缩酶，转铁蛋白， α-抗胰蛋白酶，载脂蛋白B，一些急性期基因，例如， 谷胱甘肽-S-转移酶和特异性细胞色素P450基因。 ATF 3是 由生理应激源诱导，包括：a）暴露于试剂， 四氯化碳、对乙酰氨基酚和酒精，B）机械损伤，c） 限制流向心脏的血流，和d）脑癫痫。 其 表达与细胞损伤和细胞死亡相关。 因此本 该项目的重点是ATF 3在外源性诱导的肝毒性中的作用， 人类 主要研究者假设，“扰动的 bZip合并液将导致肝功能失调。“因此， 该提案的目的是通过在人乳腺癌细胞中过表达ATF 3来验证这一假设。 使用组织特异性二元反式激活因子/反式应答因子 转基因小鼠模型。 具体目标是：1）创造个人 反式激活因子和反式应答因子转基因小鼠，2）使品系杂交， 创造生物基因小鼠，3）测试“放松管制”的后果， 在成年动物和胚胎发育过程中的“过度表达”。 肝脏特异性甲状腺素运载蛋白驱动的“反向四环素”诱导 系统将用于调节ATF 3转基因的肝表达 （用血凝素[HA]序列“标记”，其可通过 使用抗HA抗体的免疫学方法）。 为了实现这一点， 表达转甲状腺素蛋白导向四环素的低拷贝转基因小鼠 阻遏物构建体（“反式激活因子”）将被培育成低拷贝的 表达ATF 3-HA融合构建体的转基因小鼠（“transresponsers”） 来创建生物基因或双转基因品系。 后代将被用来 研究ATF 3系统在成年期以及 外源性物质引起的胎儿（肝脏）发育期。 将在成年转基因小鼠中检查ATF转基因表达。 的 需要解决的问题是增加ATF 3表达是否会增加 对外源性物质敏感。 异生物质的次优浓度 将测试药物以观察是否在生物小鼠中增加ATF 3表达 加强这些药物的效果。 异常ATF转基因的影响 还将在胚胎发育期间检查对肝功能的表达 发展

DESCRIPTION: A number of xenobiotic toxins alter expression of a subset of specific genes in the liver by first inducing expression of the gene encoding the transcription factor, ATF3, which is a bZIP (basic region-leucine zipper) protein and a member of the ATF/CREB family of transcription factors. Consensus sequences for ATF3 binding have been identified in a number of liver-specific gene promoters, specifically those thought to regulate ornithine transcarbamylase, aldolase, transferrin, alpha-antitrypsin, apolipoprotein B, some acute phase genes, e.g., glutathione-S-transferase, and specific cytochrome P450 genes. ATF3 is induced by physiological stressors including: a) exposure to agents such as carbon tetrachloride, acetaminophen and alcohol, b) mechanical injury, c) restriction of blood flow to the heart, and d) brain seizure. Its expression correlates with cellular injury and cell death. Therefore, this project focuses on the role of ATF3 in xenobiotic-induced liver toxicity in humans. The principal investigator hypothesizes that "perturbation of the bZip pool will lead to the de-regulation of liver functions." Thus, the goal of this proposal is to test the hypothesis by over-expressing ATF3 in the liver using a tissue-specific binary transactivator/transresponder transgenic mouse model. The specific aims are to: 1) create individual transactivator and transresponder transgenic mice, 2) to cross the lines to create the biogenic mice, and 3) to test the consequences of "deregulated over-expression" of ATF3 in adult animals and during embryonic development. A liver-specific, transthyretin-driven "reverse tetracycline" induction system will be used to regulate hepatic expression of an ATF3 transgene ("tagged" with a hemagglutinin [HA] sequence that is detectable by immunological methods using antiHA antibodies). To achieve this, lines of low-copy transgenic mice expressing transthyretin-directed tetracycline repressor constructs ("transactivators") will be bred to lines of low-copy transgenic mice expressing the ATF3-HA fusion construct ("transresponders") to create biogenic or double-transgenic lines. Offspring will be used to study perturbation of the ATF3 system during adulthood as well as during the period of fetal (hepatic) development by xenobiotic agents. ATF-transgene expression will be examined in adult transgenic mice. The question to be addressed is whether increased ATF3 expression increases the sensitivity to xenobiotic agents. Sub-optimal concentrations of xenobiotic agents will be tested to see if heightened ATF3 expression in biogenic mice potentiates effects of these agents. The effects of aberrant ATF-transgene expression on liver function will also be examined during embryonic development.