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内表型，可以在啮齿类动物中进行建模，并且可以反映对酒精依赖发展的易感性。针对这种表型差异而对小鼠和大鼠进行选择性育种表明，遗传因素对自愿饮酒有影响。我们使用选择性培育的高酒精偏好和低酒精偏好小鼠（分别为 HAP 和 LAP 小鼠），通过两瓶选择范式进行测量，通过大脑中的差异表达水平来识别导致酒精偏好饮酒的候选基因。我们现在建议通过使用“体素化”程序的定量逆转录酶实时 PCR (qRT-PCR) 以及定量原位杂交，在 HAP 和 LAP 小鼠的大脑中确认这些基因的差异表达，并定位大脑区域内的差异表达。然后，通过使用 RNAi 试剂治疗小鼠，将根据功能和拟议的饮酒行为中的作用来减少选定基因的表达。我们将与 INIA RNAi Core 和 Dharmacon 合作设计 siRNA 和 shRNA。这些试剂的功效和特异性将在体外进行测定。 siRNA 将使用渗透微型泵或通过含有 shRNA 的慢病毒载体的位点特异性感染来递送。我们将与 INIA Colorado Gene Array Core 合作，通过 qRT-PCR 确定靶基因下调的时间过程、持续时间和程度，并通过 qRT-PCR 和微阵列分析确定效果的特异性。一旦确认特定基因敲除，将在两瓶选择范例中测试小鼠饮酒偏好的变化。 HAP 和 LAP 小鼠还将在 INIA 小鼠动物模型核心的“戒断诱导饮酒”程序中长期接受乙醇治疗。将确定与慢性酒精暴露后自愿饮酒增加相关的大脑基因表达的变化。这些实验将系统地研究已确定的候选基因（单独或组合反映信号转导途径）在调节自愿饮酒中的作用。

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.