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中建模的内表型，这可能反映了对酒精依赖发展的易感性。对小鼠和大鼠的这种表型差异的选择性育种表明，基因对自愿饮酒有影响。我们使用了选择性饲养的高酒精偏好和低酒精偏好小鼠（分别为HAP和LAP小鼠），以两瓶选择范式进行测量，通过其在大脑中的差异表达水平来确定导致酒精偏好饮酒的候选基因。我们现在建议在HAP和LAP小鼠的大脑中，通过使用“体素化”程序的定量逆转录酶实时荧光定量PCR （qRT-PCR）和定量原位杂交，确认这些基因的差异表达，并在大脑区域内定位差异表达。选择基因的表达，根据功能和在饮酒行为中的作用进行优先排序，然后通过RNAi试剂治疗小鼠来减少。我们将与INIA RNAi Core和Dharmacon合作设计sirna和shrna。这些试剂的疗效和特异性将在体外进行检测。sirna将通过渗透微型泵或含有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。一旦特定的基因敲除被证实，将测试小鼠在两瓶选择模式下饮酒偏好的变化。在INIA小鼠动物模型核心的“戒断性饮酒”程序中，HAP和LAP小鼠也将长期用乙醇治疗。在长期酒精暴露后，大脑基因表达的变化与自愿饮酒增加相关，将被确定。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.