RNA Interference in Drosophila

果蝇中的 RNA 干扰

• 批准号: 7032257
• 负责人: Dean P. Smith
• 金额: $ 31.23万
• 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/7032257
• 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）：广泛的长期目标是从遗传学上剖析果蝇中的RNA干扰，并鉴定介导这一重要过程的分子组分。双链RNA（dsRNA）诱导有效的细胞反应，其构成对有害RNA（包括病毒、病毒复制中间体和转座因子）的基本防御。在许多系统中，dsRNA触发RNA干扰（RNAi）;与dsRNA触发物同源的转录物的显著且序列特异性的不稳定化。利用RNAi技术可以沉默正常细胞基因，这使得该技术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的确切机制尚未完全了解。基因筛选已经确定了拟南芥、脉孢菌和C.优雅的然而，这些筛选尚未饱和，仅回收可行的突变。在果蝇中筛选RNAi缺陷型突变体是不可能的，因为RNAi抑制的突变率是可变的。最近，我们通过一种新的转基因设计解决了这一难题，该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旨在阐明果蝇中RNAi的分子基础的遗传筛选奠定了基础。在具体目标1中，我们将使用FLP/FRT重组系统分离对眼睛颜色色素转运蛋白白色的RNAi抑制有缺陷和增强的突变体。这个屏幕的优点是：1.影响RNAi的突变体将容易地被鉴定为眼睛颜色突变体，2.纯合突变体克隆将被限制于复眼，因此我们将恢复在其他系统中未鉴定的RNAi所需的活基因和潜在致死基因中的突变。我们将恢复影响RNAi的抑制子和增强子突变。在特定目标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鉴定在我们的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中RNAi的理解，并拓宽我们对RNAi的总体理解。RNAi增强子突变体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更好的遗传背景。