RNA Interference in Drosophila

果蝇中的 RNA 干扰

• 批准号: 6866577
• 负责人: Dean P. Smith
• 金额: $ 31.98万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6866577
• 关键词: Drosophilidae; RNA interference; cell line; eye; gene complementation; gene induction /repression; genetic mapping; genetic recombination; genetic screening; membrane transport proteins; mutant; nucleic acid sequence; phenotype; pigments

DESCRIPTION (provided by applicant): The broad, long-term objective is to genetically dissect RNA interference in Drosophila and identify the molecular components mediating this important process. Double-stranded RNA (dsRNA) induces potent cellular responses that compose an essential defense against deleterious RNAs, including viruses, viral replication intermediates and transposable elements. In many systems, dsRNA triggers RNA interference (RNAi); a dramatic and sequence specific destabilization of transcripts homologous to the dsRNA trigger. Normal cellular genes can be silenced using RNAi making this technique an important new tool to elucidate function of orphan gene products. Although the phenomenon is remarkably similar in diverse organisms, the precise mechanisms mediating RNAi are not fully understood. Genetic screens have identified several important components required for RNAi in Arabidopsis, Neurospora and C. elegans. However, these screens have not been saturating and recovered only viable mutations. Mutant screens for RNAi defective mutants in Drosophila have not been possible because of variable penetrance of RNAi suppression. Recently we solved this difficulty with a novel transgene design that effectively silences genes in adult tissues. These studies set the groundwork for the genetic screen proposed here designed to elucidate the molecular basis for RNAi in Drosophila. In .Specific Aim 1 we will isolate mutants defective and enhanced for RNAi suppression of the eye color pigment transporter WHITE using the FLP/FRT recombination system. The advantages of this screen are: 1. mutants affecting RNAi will be easily identified as eye color mutants, 2. homozygous mutant clones will be restricted to the compound eye, therefore we will recover mutations in viable and in potentially lethal genes required for RNAi not identified in other systems, 3. We will recover both suppressor and enhancer mutations affecting RNAi. In Specific Aim 2 we will use complementation, deficiency mapping, sequence analysis of mutated candidate genes and germline transformation rescue to identify the genes responsible for the mutant phenotypes observed in our screen. Completion of the studies will enhance our understanding of RNAi in Drosophila and broaden our understanding of RNAi in general. Recovery of enhancer of RNAi mutants may provide enhanced genetic backgrounds to facilitate research on orphan gene function in Drosophila in the post-genomic era.

描述（由申请人提供）：广泛的，长期的目标是对果蝇的RNA干扰进行遗传解剖，并确定介导这一重要过程的分子成分。双链RNA （dsRNA）诱导有效的细胞反应，构成对有害RNA的必要防御，包括病毒、病毒复制中间体和转座因子。在许多系统中，dsRNA触发RNA干扰（RNAi）；与dsRNA触发器同源的转录本的戏剧性和序列特异性不稳定。利用RNAi可以使正常细胞基因沉默，使其成为阐明孤儿基因产物功能的重要新工具。尽管这种现象在不同的生物体中非常相似，但介导RNAi的确切机制尚不完全清楚。基因筛选已经确定了拟南芥、神经孢子虫和秀丽隐杆线虫中RNAi所需的几个重要成分。然而，这些筛选并没有达到饱和，只恢复了可行的突变。由于RNAi抑制的外显率不同，在果蝇中筛选RNAi缺陷突变体的突变体是不可能的。最近，我们用一种新的转基因设计解决了这个难题，这种设计有效地沉默了成人组织中的基因。这些研究为本文提出的基因筛选奠定了基础，旨在阐明果蝇RNAi的分子基础。在。我们将使用FLP/FRT重组系统分离RNAi抑制眼睛色素转运蛋白WHITE的缺陷和增强突变体。这种屏幕的优点是：1。影响RNAi的突变体很容易被识别为眼睛颜色突变体，2。纯合突变克隆将仅限于复眼，因此我们将恢复在其他系统中未发现的RNAi所需的活基因和潜在致死基因的突变。我们将恢复影响RNAi的抑制和增强突变。在Specific Aim 2中，我们将使用互补、缺陷定位、突变候选基因的序列分析和种系转化拯救来确定导致我们在筛选中观察到的突变表型的基因。这些研究的完成将增强我们对果蝇RNAi的认识，并拓宽我们对RNAi的总体认识。RNAi突变体增强子的恢复可能为后基因组时代果蝇孤儿基因功能的研究提供增强的遗传背景。