Gene Identification for Alcohol Related QTLS

描述（由申请人提供）：本修订提案的主要目标是鉴定与酒精作用相关的数量性状位点（qtl）的潜在基因。在第一个五年的资助期内，这一目标取得了相当大的进展，现在重要的新的小鼠基因组学资源和策略将被利用，这将大大加快这一进程，甚至进一步。下一个资助期的研究计划将由两个主要重点领域组成：第一个（具体目标1-2）将研究与ILS和ISS小鼠对乙醇初始敏感性相关的四个Lore qtl。先前已经对Lore2 QTL进行了大量的工作，现在将其添加到本提案中。拟议的基因鉴定工作将包括我们已经鉴定的三个改变的Lore QTL基因的持续表征和精细定位，其他新鉴定的Lore候选基因的比较测序，以及鉴定差异表达的QTL基因中的调控序列变异。loi特异性定制基因芯片已经创建，与标准小鼠芯片一起，将允许在ILS和ISS菌株之间比较所有loi基因的表达水平。第二个（Specific Aims 3-4）将是我们最近开发的一种新型计算机方法的应用，该方法用于快速识别使用C57BL/6J （B6）和DBA/2J （D2）菌株鉴定的酒精相关qtl的基因变异。这种方法代表了一种强大的新工具，将用于鉴定两个酒精相关的B6xD2 qtl的基因变异。这四个特定的目标被设计为全面的，并将用于通过测量QTL基因的蛋白质编码和调控区域来寻找潜在重要的株系间变化。这些分析将利用高通量比较DNA测序、高密度微阵列研究和利用刚刚可用的菌株特异性全基因组序列的新型计算机方法的组合。作为对上次审查的回应，具体目标5已从以前的提案中进行了重大修改并缩小了范围。这个目标现在将集中在ILS和ISS小鼠的BAC基因组文库的开发上，这将为使用转基因动物进行功能研究奠定基础。这些实验代表了该实验室先前工作的逻辑延伸，并提供了一种全面的方法来鉴定与酒精作用有关的QTL基因。

DESCRIPTION (provided by applicant): The primary goal of this revised proposal is to identify genes underlying quantitative trait loci (QTLs) related to alcohol action. Considerable progress has been made toward this goal in the first five-year funding period and now important new mouse genomics resources and strategies will be utilized that should significantly accelerate this process even further. The research plan for the coming funding period will be composed of two primary areas of focus: The first (Specific Aims 1-2) will be on four Lore QTLs related to initial sensitivity to ethanol in the ILS and ISS mice. Considerable prior work has been carried out on the Lore2 QTL which will now be added to this proposal. The proposed gene identification efforts will involve the continued characterization and fine mapping of three altered Lore QTL genes we have identified, comparative sequencing of additional, newly identified Lore candidate genes, and identification of regulatory sequence variations within differentially expressed QTL genes. Lore-specific custom gene chips have been created that together with standard mouse chips will allow the expression level of all Lore genes to be compared between ILS and ISS strains. The second (Specific Aims 3-4) will be on application of a novel in silico method we have recently developed for rapidly identifying gene variants in alcohol-related QTLs identified using the C57BL/6J (B6) and DBA/2J (D2) strains. This method represents a powerful new tool that will be used to identify gene variants in two alcohol-related B6xD2 QTLs. These four specific aims have been designed to be comprehensive, and will be used to search for potentially important interstrain changes by surveying both the protein coding and regulatory regions of QTL genes. These analyses will utilize a combination of high throughput comparative DNA sequencing, high density microarray studies and the new in silico methods that take advantage of strain-specific whole genome sequences that have just become available. In response to the last review, Specific Aim 5 has been significantly modified and reduced in scope from the previous proposal. This aim will now focus on the development of BAC genomic libraries for the ILS and ISS mice, which will set the stage for functional studies using transgenic animals. These experiments represent a logical extension of previous work from this laboratory and provide a comprehensive approach to the identification of QTL genes involved in alcohol action.