ROLE OF ALDH2--TRANSGENIC MICE CARRYING ASIAN ALDH2-2 VARIANT ALLELE

线粒体乙醛脱氢酶(ALDH2)是参与乙醛代谢的主要ALDH同工酶。已证实，导致氨基酸改变(Glu487Lys)的单核苷酸替换(G到A)会导致ALDH2活性的显性失活。我们的数据表明，重组人ALDH2变异体蛋白与小鼠ALDH2蛋白相互作用，并明显抑制小鼠ALDH2酶的活性。这种基因多态是许多亚洲人在饮酒后出现脸红反应的原因。虽然ALDH2-2等位基因已被证明对酒精中毒具有保护作用，但该酶的生理作用仍不清楚。为了进一步研究ALDH2在酒精介导的组织损伤、饮酒行为和内源和外源底物代谢中的生理作用，我们培育了携带人ALDH2变体(Haldh2-2)的转基因小鼠。目前，我们已经建立了两个独立的Haldh2转基因小鼠品系。人ALDH2蛋白在所有组织中均有表达，人ALDH2-2在转基因小鼠中的表达抑制了小鼠ALDH2酶的活性。我们还观察到，FVB/N背景系小鼠在20%乙醇处理3天后表现出恐惧、回避和逃避行为，而在乙醇暴露的转基因小鼠中这些行为变化并不明显(p&lt；0.001)。为了将表观行为变化与神经递质水平相关联，并研究ALDH2在内生代谢中的作用，用高效液相色谱法测定了各种单胺类神经递质的水平。我们的数据还表明，雄性和雌性转基因小鼠的肝脏乙醛水平都比FVB/N背景小鼠高出40%-50%(每组6只，p&lt；0.01)。我们正在用两瓶酒的选择模式来决定人们的饮酒偏好。我们到目前为止收集到的数据表明，携带Haldh2-2的转基因小鼠可以作为研究ALDH在行为、神经递质代谢和饮酒偏好中作用的有价值的模型。为了明确ALDH2的生理作用，我们利用构建的DNA载体制备了小鼠ALDH2基因缺陷的敲除小鼠。在使用敲除载体多次未能获得阳性ES细胞后，我们构建了另一种用于基因破坏的DNA载体。新的DNA计划在不久的将来注入ES细胞。所得到的ES细胞将通过Southern杂交分析筛选出阳性的ES细胞。-健康和行为、肝硬变、饮酒模式和原因、种族、分子遗传学、转基因小鼠、基因敲除小鼠

The mitochondrial aldehyde dehydrogenase (ALDH2) is the major ALDH isozyme involved in acetaldehyde metabolism. It is well established that a single nucleotide substitution (G to A) which results in the amino acid change (Glu487Lys) leads to dominant inactivation of ALDH2 activity. Our data indicated that the recombinant human ALDH2 variant protein interacted with the mouse ALDH2 protein and dominantly inhibited the activity of the mouse enzyme. The genetic polymorphism is the cause of the flushing response observed in many Asian people following alcohol intake. Although the ALDH2-2 allele has been shown to have a protective role against alcoholism, the physiological role of this enzyme is still unclear. To further examine the physiological role of ALDH2 in alcohol-mediated tissue damage, drinking behavior and metabolism of endogenous and exogenous substrates, we produced transgenic mice carrying the human ALDH2 variant (Haldh2-2). Currently, we have established two independent lines of Haldh2 transgenic mice. Human ALDH2 protein was expressed in all tissues examined and expression of human ALDH2-2 inhibited mouse ALDH2 enzyme activity in transgenic mice. We also observed that the FVB/N background strain mice showed fear, avoidance and escape behavior 3 days after treatment with 20% ethanol but these changes in behavior were not evident in the transgenic mice exposed to ethanol (p<0.001). To correlate the apparent behavioral change with levels of neurotransmitters and to study the role of ALDH2 in endobiotic metabolism, the levels of various monoamine neurotransmitters are being determined by HPLC. Our data also indicate that both male and female transgenic mice showed 40-50% higher acetaldehyde levels in the livers than the FVB/N background mice (n=6 for each group, p<0.01). We are determining the drinking preference in a two-bottle choice paradigm. Our data collected so far indicate that transgenic mice carrying the Haldh2-2 can be a valuable model to study the role of ALDH in behavior, neurotransmitter metabolism and drinking preference. In order to have clear results for the physiological roles of ALDH2, we have tried to prepare knock-out mice deficient in mouse ALDH2 gene suing a DNA vector we constructed. After repeated failures to obtain positive ES cells with the knock-out vector, we have constructed another DNA vector for gene disruption. The new DNA is planned to be injected into ES cells in the near future. The resulting ES cells will be screened for positive ES cells using Southern blot analyses. - health & behavior, cirrhosis, drinking patterns & causes, ethnicity, molecular genetics, transgenic mice, knock-out mice