ALCOHOL METABOLISM GENES IN TRANSGENIC MICE

描述（申请人提供）：酒精引起的肝脏损伤和疾病是一个重要的健康问题。肝脏中至少有三种酶活性能够将乙醇转化为乙醛，即乙醇脱氢酶（ADH）、细胞色素P450 2 E1（CYP 2 E1）和过氧化氢酶。使用天然存在的和诱导的ADH缺陷动物的结果表明I类ADH在乙醇代谢中起主要作用，IV类ADH起重要作用。I类ADH在哺乳动物的肝脏中以高水平表达。在小鼠中，单个基因Adh 1编码I类ADH，而在人类中，三个基因ADH 1A、ADH 1B和ADH 1C编码I类多肽。总体目标是鉴定控制小鼠Adh复合物成员表达表型的远端基因组元件，通过基因拷贝数调控肝脏ADH 1/CYP 2 E1比值，探讨该比值在酒精性肝损伤中的作用。BAC克隆中的连接序列将指导Adh 1在肝脏中的表达和Adh 4在胃中的表达。遗传学方法将确定Adh 1基因的重要远端控制元件，并在恒定的C57 BL/6背景下构建和使用具有不同ADH 1/CYP 2 E1比率的小鼠品系，以探索每种在酒精诱导的肝损伤中的作用。具体目的是（1）实验描绘控制Adh 1在肝脏中表达的远端序列的位置，肠和肾上腺，（2）确定在表达的Adh 1区域中是否发生染色质改变，（3）通常定义促进Adh 4适当表达表型的顺式连接序列，（4）遗传构建小鼠品系，（a）使用可用的敲除和转基因使ADH 1/CYP 2 E1比率变化8倍，（B）在肺、睾丸、精囊中过表达ADH，以及在肝脏中不存在ADH的情况下的附睾（“条件性敲除”），（c）携带用于过表达的人CYP 2 E1 BAC，以及（5）在这些遗传菌株中探索由乙醇促进的病理学。

DESCRIPTION (provided by applicant): Alcohol-induced liver damage and disease is an important health problem At least three enzyme activities in liver are capable of converting ethanol to acetaldehyde, alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1), and catalase Although CYP2E1 is inducible by chronic alcohol exposure, results using naturally occurring and induced ADH-deficient animals suggest class I ADH plays a major role in ethanol metabolism and class IV plays a significant role. Class I ADH is expressed at high levels in the liver of mammals A single gene, Adh1, encodes class I ADH in mice while three genes, ADH1A, ADH1B and ADH1C, encode class I polypeptides in humans The overall goals are to identify distal genomic dements that control the expression phenotype of members of the mouse Adh complex, and to genetically manipulate by gene copy-number the ratio of ADH1/CYP2E1 in liver to explore the role of this ratio in alcohol-induced liver damage Distal cis-linked sequence in a BAC clone will direct expression of Adh1 in liver and Adh4 in stomach No sequence directing liver expression of any human class I ADH gene is known. Genetic approaches will define the important distal control element(s) for the Adh1 gene, and construct and use mouse strains on a constant C57BL/6 background with varying ADH1/CYP2E1 ratios to explore the role of each in alcohol induced liver injury The specific aims are (1) experimentally delineate the location of the distal sequence(s) controlling expression of Adh1 in liver, intestine and adrenal using transgenic expression assays, (2) determine if chromatin alterations occur in the expressed Adh1 region, (3) generally define the cis-linked sequence that promotes Adh4 proper expression phenotype, (4) genetically construct mouse strains (a) having ADH1/CYP2E1 ratio varying 8- fold using available knockouts and transgenics, (b) overexpressing ADH in lung, testes, seminal vesicle, and epididiymis in the absence of ADH in liver (a "conditional knockout"), (c) harboring a human CYP2E1 BAC for overexpression, and (5) explore pathology promoted by ethanol in these genetic strains.