Alcohol Drinking after Modulation of Differentially Expressed Genes in HAP and LA

HAP和LA差异表达基因调节后饮酒

• 批准号: 7214266
• 负责人: Paula Hoffman
• 金额: $ 25.49万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214266
• 关键词: Lentivirus; RNA interference; alcoholic beverage consumption; alcoholism /alcohol abuse; behavioral /social science research tag; behavioral genetics; cooperative study; drug delivery systems; gene expression; gene expression profiling; genetic regulation; genetic susceptibility; in situ hybridization; laboratory mouse; microarray technology; microinjections; phenotype; polymerase chain reaction; substance abuse related behavior; transfection /expression vector; Lentivirus; RNA interference; alcoholic beverage consumption; alcoholism /alcohol abuse; behavioral /social science research tag; behavioral genetics; cooperative study; drug delivery systems; gene expression; gene expression profiling; genetic regulation; genetic susceptibility; in situ hybridization; laboratory mouse; microarray technology; microinjections; phenotype; polymerase chain reaction; substance abuse related behavior; transfection /expression vector

DESCRIPTION (provided by applicant): Voluntary alcohol consumption represents an endophenotype that can be modeled in rodents and that may reflect susceptibility to the development of alcohol dependence. Selective breeding of mice and rats for differences in this phenotype indicates a genetic influence on voluntary alcohol consumption. We have used mice selectively bred for high and low alcohol preference (HAP and LAP mice, respectively), as measured in a two-bottle choice paradigm, to identify candidate genes that contribute to alcohol preference drinking through their differential expression levels in brain. We now propose to confirm the differential expression of these genes, and localize the differential expression within brain regions, by quantitative reverse transcriptase real-time PCR (qRT-PCR) using a "voxelation" procedure, and by quantitative in situ hybridization, in brains of HAP and LAP mice. The expression of selected genes, prioritized based on function and proposed role in alcohol drinking behavior, will then be reduced by treatment of the mice with RNAi reagents. We will design siRNAs and shRNAs in collaboration with the INIA RNAi Core and Dharmacon. The efficacy and specificity of these reagents will be assayed in vitro. siRNAs will be delivered using osmotic minipumps or by site-specific infection of lentiviral vectors containing shRNAs. We will determine the time course, duration and extent of target gene down-regulation by qRT-PCR and specificity of effects by qRT-PCR and microarray analysis in collaboration with the INIA Colorado Gene Array Core. Once specific gene knockdown has been confirmed, mice will be tested for changes in alcohol preference drinking in the two-bottle choice paradigm. HAP and LAP mice will also be treated chronically with ethanol in the "withdrawal-induced drinking" procedures of the INIA Mouse Animal Models Core. Changes in brain gene expression that correlate with increases in voluntary alcohol consumption, following the chronic alcohol exposure, will be determined. These experiments will systematically investigate the role of identified candidate genes, alone and in combinations reflecting signal transduction pathways, in the modulation of voluntary alcohol consumption.

描述（由申请人提供）：自愿性饮酒代表一种可以在啮齿动物中建模的内表型，这可能反映了对酒精依赖性发展的敏感性。小鼠和大鼠的选择性育种，以差异为这种表型的差异表明对自愿饮酒的遗传影响。如在两瓶选择范式中测量的高酒精偏好（分别为HAP和LAP小鼠），我们已经使用小鼠选择性地育出来，以鉴定有助于通过其大脑中其差异表达水平饮酒的候选基因。现在，我们建议通过使用“ Voxelation”程序以及通过定量的原位杂交在HAP和LAP小鼠的大脑中，通过定量逆转录酶实时PCR（QRT-PCR（QRT-PCR）来确认这些基因的差异表达，并在大脑区域内定位差异表达。然后，根据功能和提出的在饮酒行为中提出的作用优先考虑所选基因的表达，将通过用RNAi试剂处理小鼠来降低。我们将与Inia RNAi Core和Dharmacon合作设计sirnas和Shrnas。这些试剂的功效和特异性将在体外测定。 siRNA将使用渗透微型蛋白酶或通过含有shRNA的慢病毒载体的位点特异性感染进行交付。我们将通过QRT-PCR确定目标基因下调的时间过程，持续时间和程度，以及与Inia Colorado基因阵列核心合作的QRT-PCR和微阵列分析的效应特异性。一旦确认了特定的基因敲低，将测试小鼠在两瓶选择范式中饮酒的变化。在Inia小鼠动物模型核心的“戒断诱导的饮酒”程序中，HAP和膝盖小鼠还将用乙醇长期治疗。将确定与慢性酒精暴露后自愿饮酒的增加相关的大脑基因表达的变化。这些实验将系统地研究单独的和反映信号转导途径的组合中确定的候选基因的作用，在自愿饮酒的调节中。