GENE THERAPY OF ALCOHOLISM IN A MOUSE MODEL

小鼠模型中酗酒的基因治疗

• 批准号: 2045981
• 负责人: AKIRA Y YOSHIDA
• 金额: $ 8.65万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2045981
• 关键词: alcoholism /alcohol abuse; aldehyde dehydrogenases; disease /disorder model; disease /disorder proneness /risk; enzyme activity; enzyme deficiency; gene mutation; gene therapy; genetic markers; genetically modified animals; laboratory mouse; mitochondria; polymerase chain reaction; tissue mosaicism; transfection

Individuals who lack or have severely diminished mitochondrial aldehyde dehydrogenase-2 (ALDH2) activity are at very low risk in developing alcoholism, since they cannot drink a large amount of alcohol. The deficiency is due to the atypical ALDH2 gene existing in such individuals at heterozygous ALDH2(1)/ALDH2(2) or homozygous ALDHs(2)/ALDH2(2) status. Alcoholism may be cured by introducing the nucleotide construct containing the ALDH2(2) into patients. This possibly can be tested in animal models. Mouse strain(s) associated with a high alcohol preference (such as C57BL/6J) can be changed to low alcohol preference by genetically suppressing the activity of mitochondrial ALDH. The following two studies are proposed: 1) Non-homologous integration of a mutant gene: The procedures are injections of a mutant ALDH construct(s), which is equivalent to human atypical ALDH2(2) gene, into fertilized eggs, and subsequent transplantation of the treated eggs into pseudo-pregnant mice. Transgenic mice thus produced are a sort of human heterozygous ALDH2(1)/ALDH2(2) status, and are expected to exhibit a diminished ALDH activity and a low alcohol preference. 2) Knockout the mouse mitochondrial ALDH gene targeting; The procedures include; a) transfection of mouse embryonic stem cells with a recombinant construct containing a modified ALDH gene fragment and a marker(s); b) inject transfected cells into blastocysts; c) transfer the blastocysts into pseudo-pregnant mice; and d) breeding of chimeratic offsprings to produce homozygous and heterozygous mutant strains. During the course of the experiment, incorporation of artificial mutant genes into animals is monitored by PCR-mediated DNA hybridization. Liver mitochondrial ALDH activity and alcohol preference in the original strain and the artificially produced mutant strains will be compared to verify the expectation.

线粒体醛缺乏或严重减少的个体 脱氢酶 2 (ALDH2) 活性在发育中的风险非常低 酗酒，因为他们不能大量饮酒。 这 缺陷是由于此类个体中存在非典型 ALDH2 基因 处于杂合 ALDH2(1)/ALDH2(2) 或纯合 ALDHs(2)/ALDH2(2) 状态。 酒精中毒可以通过引入含有的核苷酸构建体来治愈 ALDH2(2) 进入患者体内。 这可能可以在动物模型中进行测试。 相关小鼠品系 酒精偏好较高的（如C57BL/6J）可改为低酒精偏好 通过基因抑制线粒体活性来偏好酒精 乙醛脱氢酶。 提出以下两项研究： 1）突变基因的非同源整合：程序为 注射突变 ALDH 构建体，相当于人类 非典型 ALDH2(2) 基因进入受精卵，随后 将处理过的卵移植到假孕小鼠体内。 转基因 由此产生的小鼠是一种人类杂合 ALDH2(1)/ALDH2(2) 状态，预计会表现出 ALDH 活性降低和低 酒精偏好。 2）敲除小鼠线粒体ALDH基因靶向； 程序 包括; a) 用重组体转染小鼠胚胎干细胞 含有修饰的 ALDH 基因片段和标记的构建体； b) 将转染细胞注射至囊胚中； c) 将囊胚转移至 假怀孕小鼠； d) 培育嵌合后代以生产 纯合和杂合突变株。 在实验过程中，掺入人工突变体 通过 PCR 介导的 DNA 杂交来监测动物体内的基因。 肝 原始菌株中线粒体 ALDH 活性和酒精偏好 并将人工产生的突变株进行比较，以验证 期待。