C elegans RNAi Arrays

描述（由申请人提供）：现在大量的序列数据 增加了对大规模功能测定的需求。这里我们 建议研究RNA介导的干扰（RNAi）在 对C.优雅RNAi 影响目前太弱，不能产生可靠的无效等位基因表型。的 该提案的目标是1）鉴定RNAi超敏感突变体和2） 测试在广义筛选中使用这些突变体的可行性。我们的第一 筛选将是增加RNAi介导的沉默水平的突变 神经和肌肉特异性启动子的GFP表达。第二屏幕 将依赖于使用RNAi来拯救蠕虫从dauer发展。我们还将 检查分离的突变株对靶向dsRNA的敏感性， 在一系列组织中表达的基因。第二阶段的可行性将取决于 发现至少一个突变，增加更多的dsRNA依赖性沉默 而不是一个基因的五倍或更大的增加率。二期工程 将包括克隆和进一步表征RNAi超敏细胞 突变体和RNAi阵列的构建。 拟定商业应用： 基于功能的全基因组筛选的RNAi阵列的实施将需要 dsRNA阵列的开发，用于产生RNAi超敏反应的材料， 任何菌株背景、筛选的方案和材料，以及 处理这些测试的数据。Eon将开发、制造和分销 即用型dsRNA阵列和相关基础设施。Eon及其商业 合作伙伴还将在内部将这些阵列用于功能基因组学项目。

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.