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确定与酒精作用相关的数量性状基因座(QTL)的潜在基因。在第一个五年资助期，朝着这一目标取得了相当大的进展，现在将利用重要的新的小鼠基因组学资源和战略，这应该会进一步显着地加速这一进程。下一个资助期的研究计划将由两个主要重点领域组成：第一个领域(具体目标1-2)将是与ILS和ISS小鼠对乙醇的初始敏感性有关的四个LORE QTL。关于Lore2的QTL已经做了大量的前期工作，现在将被添加到这一提议中。建议的基因识别工作将包括对我们已经识别的三个改变的LOE QTL基因进行持续的表征和精细定位，对其他新发现的LOE候选基因进行比较测序，以及识别差异表达的QTL基因中的调控序列变异。Lore特有的定制基因芯片已经被创造出来，与标准的鼠标芯片一起，将允许比较ILS和ISS菌株之间所有LORE基因的表达水平。第二个(具体目标3-4)将是关于我们最近开发的一种新的电子计算机方法的应用，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中的基因变异。这种方法代表了一种强大的新工具，将被用于识别两个与酒精相关的B6xD2 QTL的基因变异。这四个特定的目标被设计为全面的，并将被用于通过调查QTL基因的蛋白质编码和调节区来寻找潜在的重要的菌株间变化。这些分析将利用高通量比较DNA测序、高密度微阵列研究和新的电子计算方法的组合，这些方法利用了刚刚可用的特定菌株的全基因组序列。针对上次审查，对具体目标5进行了重大修改，并缩小了先前提案的范围。这一目标现在将集中在为ILS和ISS小鼠开发BAC基因组文库，这将为使用转基因动物进行功能研究奠定基础。这些实验代表了本实验室以前工作的合理扩展，并提供了一种识别与酒精作用有关的QTL基因的综合方法。

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.