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

描述(由申请人提供)：自愿饮酒代表了一种可以在啮齿动物身上模拟的内表型，可能反映了对酒精依赖的易感性。选择性繁殖小鼠和大鼠的这种表型差异表明，基因对自愿饮酒有影响。我们使用了选择性培育的高酒精偏好和低酒精偏好的小鼠(分别为HAP和LAP小鼠)，按照两瓶选择范式的测量，以确定通过它们在大脑中的差异表达水平而导致酒精偏好饮酒的候选基因。我们现在建议确认这些基因的差异表达，并通过定量逆转录酶实时聚合酶链式反应(qRT-PCR)和定量原位杂交，在HAP和LAP小鼠的大脑中定位差异表达。根据功能和在饮酒行为中所扮演的角色，选定的基因的表达将通过RNAi试剂处理小鼠而减少。我们将与INIA RNAi Core和Dharacon合作设计siRNA和shRNA。这些试剂的有效性和特异性将在体外进行测试。SiRNAs将通过渗透性微泵或通过含有shRNAs的慢病毒载体的定点感染来传递。我们将通过qRT-PCR来确定靶基因下调的时间进程、持续时间和程度，并通过qRT-PCR和与INIA Colorado基因阵列核心合作的微阵列分析来确定效果的特异性。一旦特定的基因敲除得到确认，小鼠将在两瓶选择范例中测试饮酒偏好的变化。HAP和LAP小鼠也将在INIA小鼠动物模型核心的“戒断诱导饮酒”程序中接受长期乙醇治疗。在长期酒精暴露后，与自愿饮酒增加相关的大脑基因表达的变化将被确定。这些实验将系统地研究已识别的候选基因在调节自愿饮酒中的作用，无论是单独的还是反映信号转导途径的组合。

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.