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

描述(申请人提供)：对睡眠的重要性和睡眠障碍对健康的影响的认识日益加深，强调了对睡眠和睡眠调节的基本了解的必要性。我们的方案使用模式生物黑腹果蝇来识别当突变时会导致睡眠异常的基因。这项建议有三个具体目标：1)筛选转基因果蝇系，这些基因在突变时会导致异常的睡眠表型；2)通过检查可转座元件被精确切除的果蝇系的表型，确认表型是否属于特定的iggyBac插入；3)对特定目的确认的突变体进行额外的深入表型分析2。目前的果蝇行为分析技术依赖于红外光束断裂技术，与我们开发的基于视频的筛查分析相比，该技术在睡眠识别中的错误率较高。特别是，单红外线光束中断技术大大高估了睡眠长度，从而高估了睡眠巩固的衡量标准，使得识别睡眠长度突变变得困难。我们建议使用基于视频的方法来分析1,042条线的睡眠表型，这些线是使用iggyBac转座元件创建的。这些线是独一无二的，代表插入到以前没有发现的P元件线的基因中。可能的睡眠突变体的表型来源将被确认，我们将从分子上确认被转座元件干扰的基因(S)。一旦确认，睡眠突变者将接受额外的行为分析。这些分析包括觅食行为，测试突变对睡眠剥夺的反应，以及评估突变的昼夜节律功能。对于我们未来的目标，我们看到了两个领域的研究。如果这项概念验证研究成功，我们将为突变的睡眠表型筛选额外的转座元件插入。同时，我们建议结合分子生物学和蛋白质分析工具，通过基因组技术来表征特定基因在睡眠/清醒控制中的参与。公共卫生相关性这项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和开发睡眠障碍的新治疗方法至关重要，例如睡眠过多和失眠。

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.