Development of a Genome-wide Drosophia RNAi Resource

描述(申请人提供)：遗传学为解剖生物功能提供了最有洞察力和最强大的方法之一，果蝇已成为此类分析的主要模式生物，其中许多已被探索与人类健康相关的过程。现在，RNAi的基因失活正在彻底改变遗传方法。通过实现极快的全基因组筛选，全基因组RNAi(DmRNAi)资源将极大地加速果蝇的遗传学研究。RNAi方法将立即在功能和分子数据之间建立联系。此外，通过建立用于这些分析的双链RNA参考集合，在研究各种问题的众多实验室中收集的数据可以被组合成一个通用的功能数据库，这将鼓励发现新的联系。在成功构建代表果蝇最保守基因的不完整双链RNA文库的基础上，我们将首先建立一个完整的文库。然后，我们将制定方法，以降低成本、促进分配、鼓励使用并增加方法的灵活性。我们将探索在全基因组范围内以可接受的成本结构制备RNA的方法。最后，将探索一种标记策略，作为一种方法，它应该允许通过汇集DNA模板来压缩复杂的文库，从而可以存储和从5个试管中检索包含14,000个构建体的文库。文库本身和拟议的技术进步将通过提供覆盖整个果蝇基因组的完整集合，促进将数据积累到通用数据库中，降低这些研究的成本，并将启动全基因组RNAi筛选的技术障碍降至最低，从而在广泛获得RNAi使基因失活的力量方面发挥带头作用。根据这笔资金，将有三种产品商业化：(1)代表所有果蝇基因的DNA构建物，用于生成RNA；(2)从RNAi‘Ready’的DNA构建物获得的RNA；以及(3)基因组标签策略下可用于筛选整个基因组的一系列池。

DESCRIPTION (provided by applicant): Genetics has provided one of the most discerning and powerful approaches to the dissection of biological function and Drosophila has served as a major model organism for such analyses, many of which have been probed processes relevant to human health. Now gene inactivation by RNAi is revolutionizing genetic approaches. By making possible extremely rapid whole genome screens, a Genome-wide Drosophila RNAi (dmRNAi) resource will tremendously accelerate genetic studies in Drosophila. The RNAi approach will immediately establish a link between function and molecular data. Furthermore, by establishing a reference collection of double stranded RNAs for these analyses, data gathered in numerous laboratories studying diverse problems can be assembled into a common functional data base that will encourage discovery of novel connections. Building on successful construction of an incomplete library of double stranded RNAs representing most conserved genes of Drosophila, we will first establish a complete library. We will then develop approaches to reduce cost, facilitate distribution, encourage use and increase the flexibility of the methods. We will explore methods for the preparation of RNA on a genome-wide scale at an accessible cost structure. Finally, a tagging strategy will be explored as an approach that should allow compression of complex libraries by allowing pooling of DNA templates such that a library of 14,000 constructs can be stored and retrieved from 5 tubes. The library itself and the proposed technical advances will lead the way in making the power of gene inactivation by RNAi available widely by providing a complete collection covering the entire Drosophila genome, facilitating the accrual of data into a common data base, reducing the cost of these studies, and minimizing the technical impediments to initiating genome-wide RNAi screens. Three products will be commercialized under this funding: (1) DNA constructs representing all of the Drosophila genes for use in generating RNA, (2) RNA from the DNA constructs that are RNAi 'ready', and (3) a series of pools available under the genome tagging strategy useful in screening the entire genome.