IDENTIFICATION OF GENES INDUCED OR REPRESSED BY CHRONIC ALCOHOL

拟议中的项目采用了一种新的方法来识别 在调节小鼠酒精戒断严重程度中的重要性 鉴定慢性酒精暴露过程中表达和调控的mRNAs。 潜在的假设是，基因表达的变化可能会 在长期接触乙醇后发生在大脑中，任何基因 其表达受慢性乙醇的影响是一个很好的候选者 在酒精神经适应中具有重要的功能作用。基因 长期接触酒精可能会增加戒断风险，或者 防止提款的专家保护。我们建议将小说 重要的候选基因，对潜在角色没有偏见，使用 差异显示技术。 近交系WSP-2小鼠吸入使身体依赖， 从暴露的和对照的小鼠的大脑中提取的蛋白质将通过 差异显示。依赖和控制的RNA将与 改良的寡聚脱氧核糖核糖核酸(OGO-DT)引物，反转录。反转录本 用随机引物子集进行聚合酶链式反应，扩增出cDNAs序列； 这些产品被贴上标签，并在DNA测序凝胶上分离。通过比较 不同组别的带型，具有相似带型 对照样本和处理样本中的强度都将被忽略， 使多个基因产品有差别地展示(即， 上调或下调)可能是由于乙醇对 基因表达。

The proposed project takes a novel approach to identifying the genes of importance in modulating the severity of alcohol withdrawal in mice by identifying mRNAs expressed and regulated during chronic ethanol exposure. The underlying hypothesis is that changes in gene expression are likely to occur in the brain after chronic exposure to ethanol, and that any gene whose expression is affected by chronic ethanol is a good candidate for having a functionally important role in ethanol neuroadaptation. Genes regulated by chronic ethanol exposure might promote withdrawal risk, or expert protection against withdrawal. We propose to identify novel candidate genes of importance, without bias about potential roles, using the technique of differential display. Inbred WSP-2 mice will be made physically dependent by inhalation, and mRNA from the brains of exposed and control mice will be analyzed by differential display. Dependent and control RNAs will be hybridized to modified oligo-dT primers and reverse transcribed. The reverse transcribed cDNA sequences will be amplified by PCR with subsets of random primers, and the products labeled and separated on a DNA sequencing gel. By comparing the banding patterns for the different groups, bands with similar intensities in both the control and treated samples will be ignored, leaving a number of gene products differentially displayed (i.e., either up- or down-regulated) presumably as a result of an effect of ethanol on gene expression.