TRiP resources for modeling human disease

项目总结/摘要 该申请要求继续资助TRiP，这是一个非常成功的果蝇体内功能研究。 哈佛医学院基因组学平台。到目前为止，我们已经产生了超过17，000个RNAi， CRISPR为研究界提供的苍蝇种群。我们建议扩大TRiP资源的多功能和 用于基因激活、抑制和基因组工程的转化转基因工具。这个的目标 资源，集中在3017个果蝇基因，这些基因与已知或怀疑与 （1）产生用于RNAi的shRNA果蝇原种，其解决集合中剩余的缺口， 例如尚未涵盖的基因或被社区鉴定为无效的试剂;（2）产生sgRNA 用于CRISPR敲除（CRISPRko）和CRISPR激活（CRISPRa）的储备;（3）构建试剂工具包 该联合收割机GAL 4/UAS和第二个二元表达系统（莱克萨/lexAop或QF/QUAS）组合以促进RNAi 和/或CRISPR控制两种不同组织中的基因表达;和（5）评估整体 动物以及组织特异性功能丧失（LOF）和功能获得（GOF）表型与 在我们的“RSVP Plus”中，我们将为您提供新品系的信息，并为您提供有关单个RNAi和sgRNA品系质量的信息。 表型数据库这些努力的结果将是一个巨大的苍蝇资源，分配给 社区由布卢明顿果蝇库存中心，这将使研究人员很容易击倒， 敲除或激活该集合所涵盖的基因。当与广泛的GAL 4线组合时， 我们提出的新的莱克萨/QF系的集合，这个遗传工具包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.