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

描述（由申请人提供）：越来越多的人认识到睡眠的重要性和睡眠障碍对健康的影响，强调了对睡眠和睡眠调节有基本了解的必要性。我们的建议使用模式生物黑腹果蝇来识别突变时导致异常睡眠的基因。该提案有三个具体目的：1)筛选通过piggyBac-element介导的突变基因创建的转基因果蝇，这些基因在突变时导致异常睡眠表型；2)通过检查精确切除转座元件的蝇系的表型，确认表型分配到特定的piggyBac插入；3)对Specific Aim 2中确认的突变体进行进一步深入的表型分析。目前的果蝇行为分析技术依赖于红外波束断裂技术，与我们开发的基于视频的筛选分析相比，该技术在睡眠识别方面的错误率很高。特别是，单红外波束断裂技术大大超过了估计的睡眠时间，因此测量睡眠巩固，使得识别睡眠时间突变变得困难。我们建议使用基于视频的方法来分析使用piggyBac转座元件创建的1,042个系的睡眠表型。这些系是独一无二的，并且代表了插入到先前生成的p元素系中没有发现的基因中。piggyBac插入作为假定睡眠突变的表型来源的分配将被确认，我们将从分子上确认被转座元件破坏的基因。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。我们看到了两个研究领域的未来目标。如果这个概念证明研究是成功的，我们将筛选额外的转座因子插入突变睡眠表型。同时，我们建议使用分子生物学和蛋白质分析工具的组合，通过基因组技术来描述在睡眠/觉醒控制中确定的特定基因的参与。与公共卫生的关系本提案将探讨睡眠的遗传基础。了解睡眠的遗传学对我们理解睡眠的功能和开发睡眠障碍的新疗法（如嗜睡和失眠）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.