ALCOHOL METABOLISM GENES IN TRANSGENIC MICE

描述(申请人提供)：酒精诱导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，酒精脱氢酶(ADH)，细胞色素P450 2E1(CYP2E1)和过氧化氢酶尽管慢性酒精暴露可诱导CYP2E1，但使用自然发生和诱导的ADH缺乏的动物的结果表明，I类ADH在乙醇代谢中起主要作用，IV类起重要作用。第I类ADH在哺乳动物肝脏中高水平表达单个基因Adh1在小鼠中编码第I类ADH，而三个基因ADH1A、ADH1B和ADH1C在人类中编码第I类多肽总的目标是确定控制小鼠ADH复合体成员表达表型的远端基因组元件，并通过基因拷贝数从遗传学上操纵ADH1/CYP2E1在肝脏中的比例，以探讨这一比例在酒精性肝损伤中的作用。遗传学方法将定义Adh1基因的重要远端调控元件(S)，并在恒定的C57BL/6背景下构建和使用不同的Adh1/CYP2E1比率的小鼠品系，以探索每种基因在酒精性肝损伤中的作用。具体目的是(1)通过转基因表达分析实验描述控制Adh1在肝脏、肠道和肾上腺表达的远端序列(S)的位置，(2)确定在表达的Adh1区是否发生染色质变化，(3)总体上定义促进Adh4正确表达表型的顺式连锁序列。(4)利用现有的基因敲除和转基因技术，从基因上构建ADH1/CYP2E1比值变化8倍的小鼠品系，(B)在肝脏中不存在ADH的情况下，ADH在肺、睾丸、精囊和附属膜中过表达(条件基因敲除)，(C)携带人CYP2E1BAC进行过表达，以及(5)探索酒精在这些遗传菌株中促进的病理学。

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.