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在表型，可以在啮齿动物中建模，并可能反映对酒精依赖发展的易感性。对小鼠和大鼠进行选择性繁殖，以寻找这种表型的差异，表明自愿饮酒受到遗传影响。我们使用了选择性地为高酒精偏好和低酒精偏好（分别为HAP和HAP小鼠）繁殖的小鼠，如在两瓶选择范例中所测量的，以通过其在大脑中的差异表达水平来识别有助于酒精偏好饮酒的候选基因。我们现在建议，以确认这些基因的差异表达，并本地化的差异表达的大脑区域内，通过定量逆转录酶实时PCR（qRT-PCR）使用的“voxetamine”程序，并通过定量原位杂交，在大脑中的HAP和HAP小鼠。然后，基于功能和在饮酒行为中的拟议作用而优先考虑的所选基因的表达将通过用RNAi试剂处理小鼠来减少。我们将与INIA RNAi Core和Dharmacon合作设计siRNA和shRNA。将在体外测定这些试剂的有效性和特异性。siRNA将使用渗透微泵或通过含有shRNA的慢病毒载体的位点特异性感染来递送。我们将与INIA科罗拉多基因阵列核心合作，通过qRT-PCR和微阵列分析确定靶基因下调的时程、持续时间和程度，以及效应的特异性。一旦特定的基因敲除得到证实，将测试小鼠在两瓶选择范例中的酒精偏好饮酒的变化。在INIA小鼠动物模型核心的“戒断诱导饮酒”程序中，还将用乙醇长期处理HAP和HAP小鼠。将确定与慢性酒精暴露后自愿饮酒量增加相关的脑基因表达变化。这些实验将系统地研究所确定的候选基因的作用，单独和组合反映信号转导途径，在自愿饮酒的调制。

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.