DEVELOPMENT OF CONGENIC STRAINS/IDENTIFICATION OF THE GENE UNDERLYING LORE2

同源菌株的开发/LORE2 基础基因的鉴定

• 批准号: 6299164
• 负责人: JAMES M SIKELA
• 金额: $ 15万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6299164
• 关键词: alcoholism /alcohol abuse; animal breeding; animal genetic material tag; behavioral /social science research tag; behavioral genetics; disease /disorder model; genetic mapping; genetic markers; genetic strain; inbreeding; laboratory mouse; model design /development; quantitative trait loci; sleep disorders

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.)

这一部分的目标是确定小鼠基因的基础 Lore 2，位于小鼠2号染色体上的酒精敏感性QTL， 提供同类小鼠品系，以用于 科罗拉多大学酒精研究中心（CU ARC）。的重点 ISS和ILS品系的小鼠在 乙醇给药后翻正反射丧失（LORR）的时间长度。 这种差异LORR已被证明与几种 不同的染色体区域（QTL），每个区域都解释了一部分 差传说2解释了睡眠中约14.2%的遗传变异 时间（近交系之间的25分钟差异）。该QTL已被 在RI（p <0.001）和F2中均检测到 （lodmax=6.6），并已使用定向基因组选择得到证实 方法论。Lore 2位于2号染色体的85 cM附近（l-lod-support 间隔为78-95 cM。组件3将同时执行鼠标 将Lore 2间隔缩小到1 cM以下的育种努力 (Dr.约翰逊）和基因鉴定策略，将候选基因 序列差异的间隔（Sikela博士）。我们还将 与其他ARC研究者互动，以建立功能确认 该组分鉴定的基因，并提供同源菌株 对于其他组件。小鼠遗传组件将执行两项 不同的功能。首先，将构建一系列同源性 在ILS背景上携带每个短睡眠QTL（互补的 同源基因，ISS背景上的L等位基因，正在由RO 1开发）。 其次，利用标记将Lore 2 QTL定位到1cM或更小 跨越2号染色体上的78-95 cM区间。两个一般目标都使用 速度同源的方法，以回交的国际空间站ILS和互惠 以及SSLP标记的分型。三轮回传将是 使用速度同源分析的辅助进行， 如果小鼠在第四轮和随后的 完成了七轮的回传。将评估小鼠的 在染色体2上的这段时间内发生重组事件，将Lore 2定位到 大约1cM，概率大于50%。用于基因 识别，主要战略将是迅速利用 与EST/基因和基因相关的新兴小鼠和人类基因组资源 地图通过使用这些资源，小鼠的cDNAs，位于 将确定Lore 2间隔并用于基于PCR的直接测序 ILS和ISS小鼠之间的比较。一旦序列差异 将通过适当的功能测定和其他方法进行测试。 通过与其他ARC实验室的合作进行验证方法。 由此产生的Lore 2基因的人类对应物将是一个候选者， 导致了酗酒的遗传倾向(For那些 不熟悉专门的遗传学术语，一个词汇已经被 如表1所示）。