TRiP resources for modeling human disease

项目摘要/摘要 这项申请要求继续资助行程，在体内非常成功的果蝇功能 哈佛医学院的基因组学平台。到目前为止，我们已经生成了超过17,000个RNAi和 CRISPR为研究界提供飞行库存。我们建议扩大通用型和多功能型的出行资源。 用于基因激活、抑制和基因组工程的变革性转基因工具。这样做的目的是 资源，集中在3017个与已知或怀疑与之相关的基因同源的苍蝇基因上 人类疾病，将：(1)产生用于RNAi的shRNA苍蝇种群，以解决收集中的剩余缺口， 例如尚未被覆盖的基因或被社区识别为无效的试剂；(2)产生sgRNA CRISPR基因敲除(CRISPRko)和CRISPR激活(CRISPRa)的库存；(3)建立试剂工具包 将GAL4/UAS和第二个二元表达系统(LexA/LexAop或QF/Quas)相结合以促进RNAi 和/或CRISPR对两个不同组织中基因表达的独立控制；以及(5)整体评估 与以下相关的动物和组织特异性功能丧失(LOF)和功能获得(GOF)表型 关于RNAi和sgRNA品系在我们的“RSVP Plus”中的质量的新品系和辅助信息 表型数据库。这些努力的结果将是巨大的苍蝇种群资源，分配给 布鲁明顿果蝇种群中心的社区，这将使研究人员能够轻松地拆毁， 敲除或激活收集到的基因。当与宽阵列的GAL4线相结合时，以及 我们提出的新的LexA/QF系集合，这个遗传工具箱将允许研究人员调节基因 在任何给定的阶段和同时在多个组织中表达。

PROJECT SUMMARY / ABSTRACT This application requests continued funding for the TRiP, a highly successful in vivo Drosophila functional genomics platform at Harvard Medical School. To date we have generated more than 17,000 RNAi and CRISPR fly stocks for the research community. We propose to expand the TRiP resource of versatile and transformative transgenic tools for gene activation, repression and genome engineering. The goals of this resource, focused on the 3017 fly genes orthologous to genes known or suspected to be associated with human diseases, are to: (1) generate shRNA fly stocks for RNAi that address remaining gaps in the collection, such as genes not yet covered or reagents identified by the community as ineffective; (2) generate sgRNA stocks for CRISPR knock out (CRISPRko) and CRISPR activation (CRISPRa); (3) Build a toolkit of reagents that combine GAL4/UAS and a second binary expression system (LexA/lexAop or QF/QUAS) to facilitate RNAi and/or CRISPR control of gene expression in two different tissues independently; and (5) evaluate whole animal as well as tissue-specific loss-of-function (LOF) and gain-of-function (GOF) phenotypes associated with the new lines and curate information on the quality of individual RNAi and sgRNA lines in our “RSVP Plus” phenotype database. The result of these efforts will be a tremendous resource of fly stocks, distributed to the community by the Bloomington Drosophila Stock Center, which will allow researchers to easily knock down, knock out, or activate genes covered by the collection. When combined with the wide array of GAL4 lines, and our proposed new collection of LexA/QF lines, this genetic toolkit will allow researchers to modulate gene expression in any given stage and in multiple tissues simultaneously.