A Novel-Video Based Strategy for Identifying Sleep Mutants in Drosophila

描述（由申请人提供）：对睡眠的重要性和睡眠障碍对健康的影响的日益认识强调了对睡眠和睡眠调节的基本理解的必要性。我们的建议使用模式生物黑腹果蝇（Drosophila melanogaster）来识别突变时导致异常睡眠的基因。该建议有三个具体目的：1）筛选通过piggyBac元件介导的突变产生的转基因果蝇品系，以寻找突变时引起异常睡眠表型的基因; 2）通过检查转座元件已被精确切除的果蝇品系的表型，确认表型归属于特定的piggyBac插入; 3）对特定目标2中确认的突变体进行额外的深入表型分析。目前的果蝇行为分析技术依赖于红外光束中断技术，与我们开发的基于视频的筛选分析相比，该技术在睡眠识别中具有较高的错误率。特别地，单红外光束中断技术显著地高估了睡眠时间，因此高估了睡眠巩固的测量，使得难以识别睡眠时间突变体。我们建议使用基于视频的方法来分析使用piggyBac转座因子创建的1，042行的睡眠表型。这些品系是独特的，代表插入到先前未发现的P-元件品系的基因中。piggyBac插入作为假定的睡眠突变体的表型来源的分配将得到证实，我们将从分子上证实转座因子破坏的基因。一旦确认，睡眠突变体将接受额外的行为分析。这些分析包括摄食行为，测试突变体对睡眠剥夺的反应和评估突变体的昼夜节律功能。我们看到两个领域的研究，我们的未来目标。如果这一概念验证研究成功，我们将筛选额外的转座因子插入突变睡眠表型。同时，我们建议的特征参与特定的基因识别的睡眠/觉醒控制基因组技术，使用分子生物学和蛋白质分析工具的组合。公共卫生相关性该提案将解决睡眠的遗传基础。了解睡眠的遗传学对于我们理解睡眠的功能以及开发睡眠障碍（如嗜睡和失眠）的新疗法至关重要。

DESCRIPTION (provided by applicant): An increasing appreciation of the importance of sleep and the impact of sleep disorders on health has underlined the need for a basic understanding of sleep and sleep regulation. Our proposal uses the model organism Drosophila melanogaster to identify genes that when mutated lead to abnormal sleep. This proposal has three specific aims: 1) To screen lines of transgenic Drosophila created through piggyBac-element mediated mutagenesis for genes which, when mutated, cause an abnormal sleep phenotype; 2) To confirm the assignment of the phenotype to a particular piggyBac insertion by examining the phenotype of fly lines in which the transposable element has been precisely excised; 3) To perform additional in-depth phenotypic analysis of mutants confirmed in Specific Aim 2. Present techniques for behavioral analysis of Drosophila rely on infrared beam break technology, which has a high error rate in sleep identification in comparison to the video-based screening analysis we have developed. In particular, single infrared beam break technology significantly over estimates sleep bout length and hence the measure of sleep consolidation, making identification of sleep bout length mutants difficult. We propose using video-based methods to analyze the sleep phenotypes of 1,042 lines that were created using the piggyBac transposable element. These lines are unique and represent insertions into genes that are not found previously generated P-element lines. The assignment of the piggyBac insertion as the source of the phenotype of putative sleep mutants will be confirmed and we will molecularly confirm the gene(s) disrupted by the transposable element. Upon confirmation sleep mutants will be subjected to additional behavioral analyses. These analyses include feeding behavior, testing the mutant response to sleep deprivation and assessing of mutant circadian function. We see two areas of research for our future goals. If this proof of concept study is successful, we will screen the additional transposable element insertions for mutant sleep phenotypes. Concurrently, we propose to characterize the involvement of the specific genes identified in sleep/wake control by genomic techniques using a combination of molecular biological and protein analysis tools. PUBLIC HEALTH RELEVANCE This proposal will address the genetic basis for sleep. Understanding the genetics of sleep is crucial to our understanding of the functions sleep fulfills and the development of novel treatments for sleep disorders, such as hypersomnia and insomnia.