DEVELOPMENT OF CONGENIC STRAINS/IDENTIFICATION OF THE GENE UNDERLYING LORE2

这一部分的目标是确定小鼠的基因 位于小鼠2号染色体上的酒精敏感性QTL Lore2 提供同源小鼠品系，以供使用的其他成分 科罗拉多大学酒精研究中心(CU ARC)。我们的重点是 工作是ISS和ILS品系的小鼠，它们在 乙醇给药后的翻正反射丧失长度(LORR)。 这种差异LORR已被证明与几个 不同的染色体区域(QTL)，每个区域解释 不同。传说2解释了睡眠中约14.2%的遗传差异 时间(自交系之间的差异为25分钟)。该QTL已被 在RIS(p<0.001)和F2‘S中都检测到 (lowmax=6.6)，并已通过定向基因组选择得到证实 方法论。Lore2位于2号染色体85 cM附近(L-LOD-Support 间隔78-95厘米。组件3将同时执行鼠标 将Lore2间隔缩小到1 cm以下的育种努力 (约翰逊博士)和候选基因的基因识别策略 在序列差异的间隔中(西克拉博士)。我们还将 与其他ARC调查人员互动以确定功能确认 由该成分鉴定的基因(S)，并提供同源菌株 用于其他组件。小鼠遗传组件将执行两个 不同的功能。首先，将构建一系列同源基因 在ILS背景上携带每个短睡眠QTL(互补 遗传，L在国际空间站背景下的等位基因，正在由一个RO1)开发。 其次，利用标记将Lore2 QTL定位到1 cM或更小 在2号染色体上跨越78-95厘米的间隔。这两个一般目标使用 ISS与ILS回交及其正反交的快速同源途径 同时进行SSLP标记分型。三轮回交将是 在快速同源分析的帮助下执行，然后 第四轮及后续实验小鼠的表型鉴定 又完成了七轮回交。小鼠将被评估为 2号染色体上这个区间的重组事件将Lore2定位到 大约1厘米，概率比50%更高。对于基因 识别，主要战略将是迅速利用 新兴的与EST/基因和基因相关的小鼠和人类基因组资源 地图。通过使用这些资源，位于 将识别Lore2区间并将其用于基于PCR的直接序列 ILS小鼠与ISS小鼠的比较。一旦序列差异被 获得的它们将通过适当的功能分析和其他 验证性方法通过与其他ARC实验室的合作进行。 由此产生的Lore2基因的人类对应体将是 导致酒精中毒的遗传易感性。(适用于 由于不熟悉专门的遗传学术语，一部词典 如表1所示。)

The goals of this component are to identify the mouse gene underlying Lore2, a QTL for alcohol sensitivity located on mouse chromosome 2, and to provide congenic mouse strains for the use of other components of the University of Colorado Alcohol Research Center (CU ARC). The focus of our work is the ISS and ILS strains of mice which differ markedly in the length of loss of righting reflex (LORR) after ethanol administration. This differential LORR has been shown to be associated with several distinct chromosomal regions (QTLs), each of which explains part of the difference. Lore 2 explains about 14.2 % of the genetic variance in sleep time (a 25 minute difference between the inbreds). This QTL has been detected has been detected both in RIs (p less than 0.001) and in F2's (lodmax=6.6) and has been confirmed using directed genome selection methodologies. Lore2 is located near 85 cM of chromosome 2 (l-lod-support interval is from 78-95 cM. Component 3 will simultaneously carry out mouse breeding efforts that will narrow the Lore2 interval to less than 1 cM (Dr. Johnson) and gene identification strategies that will candidate genes in the interval for sequence differences (Dr. Sikela). We will also interact with other ARC investigators to establish functional confirmation of the gene(s) identified by this component and provide congenic strains for the other components. The mouse genetic component will perform two different functions. First, a series of congenics will be constructed carrying each short-sleep QTL on the ILS background (the complementary congenics, L alleles on an ISS background, are being developed by an RO1). Second, the Lore2 QTL will be localized to 1cM or less using markers spanning the 78-95 cM interval on chromosome 2. Both general aims use speed congenic approaches to backcrosses of ISS to ILS and the reciprocal together with typing of SSLP markers. Three rounds of backcrosses will be performed using the assistance of a speed congenic analysis followed by assessment of phenotype if mice in the fourth round and subsequent completion of seven more rounds of backcrosses. Mice will be assessed for recombination events in this interval on chromosome 2 to localize Lore2 to approximately 1cM at better than a 50% probability. For gene identification, the primary strategy will be to utilize the rapidly emerging mouse and human genome resource relating to ESTs/genes and gene maps. Through the use of these resources, mouse cDNAs that lie in the Lore2 interval will be identified and used for PCR-based direct sequence comparisons between ILS and ISS mice. Once sequence differences are obtained they will be tested by appropriate functional assays and other confirmatory methods though collaborations with other ARC laboratories. The human counterpart of the resulting Lore2 gene will be a candidate for contributing to genetic predisposition to alcoholism. (For those unfamiliar with the specialized genetics terminology, a lexicon has been provided as Table 1.)