C elegans RNAi Arrays

描述(申请人提供)：目前海量的序列数据 可获得性增加了对大规模功能分析的需求。在这里，我们 建议研究RNA介导的干扰(RNAi)在 对线虫基因缺失的影响进行全基因组调查。RNAi 目前影响太弱，无法产生可靠的零等位基因表型。这个 这项建议的目标是1)鉴定RNAi超敏突变体和2) 测试使用这些突变体进行一般筛选的可行性。我们的第一次 筛选会增加RNAi介导的沉默水平的突变 神经和肌肉特异性启动子的绿色荧光蛋白表达。第二个屏幕 将依赖于使用RNAi从Dauer发育中拯救蠕虫。我们还将 检测分离的突变菌株对靶标dsRNAs的敏感性 基因在一系列组织中表达。第二阶段的可行性将取决于 发现至少一个突变增加了依赖dsRNA的更多 而不是一个外显率增加五倍或更多的基因。第二阶段工程 将包括RNAi超敏反应的克隆和进一步鉴定 突变体和RNAi阵列的构建。 建议的商业应用： 实现基于功能的全基因组筛选的RNAi阵列将需要 产生RNAi超敏反应的dsRNA阵列材料的研究进展 任何菌株背景、筛查方案和材料，以及 处理这些测试的数据。意昂将开发、制造和分销 现成的dsRNA阵列和相关的基础架构。意昂及其商业化 合作伙伴还将在内部将这些阵列用于功能基因组学计划。

DESCRIPTION (provided by applicant): The vast amount of sequence data now available has increased the demand for large-scale functional assays. Here, we propose to investigate the use of RNA-mediated interference (RNAi) in performing genome-wide surveys for the effects of gene loss in C. elegans. RNAi effects are currently too weak to produce reliable null allele phenocopies. The goals of this proposal are 1) to identify RNAi hypersensitive mutants and 2) to test the feasibility of using these mutants in generalized screening. Our first screen will be for mutations that increase the level of RNAi-mediated silencing of GFP expression from nerve and muscle-specific promoters. The second screen will rely on using RNAi to rescue worms from dauer development. We will also examine the sensitivity of the isolated mutant strains to dsRNAs for target genes expressed in a range of tissues. Phase II feasibility will depend on finding at least one mutation that increases dsRNA-dependent silencing of more than one gene with a five-fold or greater increase in penetrance. Phase II work will include cloning and further characterization of the RNAi hypersensitive mutants and construction of an RNAi array. PROPOSED COMMERCIAL APPLICATION: Implementation of RNAi arrays for function-based genome-wide screens will require the development of dsRNA arrays, materials for producing RNAi hypersensitivity in any strain background, protocols and materials for the screens, and informatics for handling the data from these tests. Eon will develop, manufacture and distribute ready-to-use dsRNA arrays and the associated infrastructure. Eon and its commercial partners will also employ these arrays in-house for functional genomics programs.