New players in immune function: identification through RNAi and micro-RNA screens

描述（由申请人提供）：尽管基因组计划在列出每个基因方面取得了成功，但对于大多数基因在复杂的生物过程中如何发挥作用，仍有很多需要了解的地方。在哺乳动物中尤其如此，在哺乳动物中，高通量遗传方法要么不可行，要么非常耗时。事实证明，细胞系中的RNA干扰（RNAi）筛选在这方面非常有用；特别是，利用生物选择能力的RNAi和微rna （miRNA）筛选在未来具有巨大的潜力。在本申请中，我们请求资助建立RNAi/ miRNA筛选设施，以解决RFA五个研究主题中的两个。(i)该设施将极大地增强我们“应用基因组学和其他高通量技术”研究免疫功能的能力，提供高通量仪器和RNAi/ miRNA文库，用于以下概述的大规模RNAi和miRNA筛选。（ii）作为一项旨在在拟议的3年项目期结束时实现自我维持的资源，该设施将通过提供RNAi筛选设计的仪器、试剂和建议，为圣地亚哥的“当地生物医学研究界注入活力和活力”，我们相信这将促进新的发现、合作和跨学科研究。该设施将设在拉霍亚过敏与免疫学研究所（LIAI），最初将由LIAI和斯克里普斯研究所（TSRI）的研究人员用于研究对免疫学至关重要的基本问题。在项目1 （LIAI）中，我们将使用全基因组siRNA筛选来识别细胞对双链RNA （ds）反应中缺失的参与者，并使用聚焦慢病毒shRNA筛选来识别启动对外来微生物dsDNAs反应的未知dna结合蛋白。在项目2 （TSRI）中，我们将筛选一个表达mirna的慢病毒的综合文库，以研究它们在体内消除B细胞耐受性的能力。在项目3 （LIAI）中，我们将使用这个miRNA文库，以及一个针对转录因子和染色质相关蛋白的慢病毒shRNA文库，来确定影响CD8 T细胞决定表达效应细胞或记忆细胞命运标记的候选物。在项目4 （LIAI）中，我们将使用这个慢病毒shRNA文库来鉴定影响iNKT细胞表达细胞因子白介素-4 （IL-4）和干扰素-3 （IFN-3）能力的转录因子和染色质相关蛋白，首先在培养的人类细胞系中，然后在体内进行shRNA筛选。总之，这些研究使用了最先进的方法——基于细胞的筛选格式，具有多个同时读数，以及复杂的汇集筛选技术，利用体内强大的生物选择——来扩展我们对不同细胞背景下免疫功能的理解。

DESCRIPTION (provided by applicant): Despite the success of genome projects at listing every gene, there is still a great deal to be understood about how the large majority of genes function in complex biological processes. This is especially true in mammals, where high-throughput genetic approaches are either not feasible or are very time-consuming. RNA interference (RNAi) screens in cell lines have proven extremely useful in this regard; in particular, RNAi and micro-RNA (miRNA) screens that harness the power of biological selection in vivo have enormous potential for the future. In this application, we request funding to build an RNAi/ miRNA screening facility that addresses two of the five research themes of the RFA. (i) The facility will greatly enhance our ability to "apply genomics and other high-throughput technologies" to studies of immune function, by providing the high-throughput instruments and RNAi/ miRNA libraries needed for the large-scale RNAi and miRNA screens outlined below. (ii) As a resource intended to be self-sustaining by the end of the proposed 3-year project period, the facility will "reinvigorate and energise the local biomedical research community" in San Diego by providing instrumentation, reagents and advice on RNAi screen design that we believe will catalyze new discoveries, collaborations and cross-disciplinary research. The facility will be based at the La Jolla Institute for Allergy & Immunology (LIAI) and will be used initially by researchers at the LIAI and The Scripps Research Institute (TSRI) to investigate questions of fundamental importance to immunology. In Project 1 (LIAI), we will use a genome-wide siRNA screen to identify missing players in the cellular response to double-stranded (ds) RNA, and a focused lentiviral shRNA screen to identify the unknown DNA-binding protein(s) that initiate responses to foreign microbial dsDNAs. In Project 2 (TSRI), we will screen a comprehensive library of miRNA-expressing lentiviruses for their ability to abrogate B cell tolerance in vivo. In Project 3 (LIAI), we will use this miRNA library, as well as a focused lentiviral shRNA library directed against transcription factors and chromatin-associated proteins, to identify candidates that influence the decision of CD8 T cells to express markers of effector or memory cell fate. In Project 4 (LIAI), we will use this lentiviral shRNA library to identify transcription factors and chromatin-associated proteins that influence the ability of iNKT cells to express the cytokines interleukin-4 (IL-4) and interferon-3 (IFN-3), first in cultured human cell lines and later in pooled shRNA screens in vivo. Together these studies use state-of-the art approaches -- cell-based screening formats with multiple simultaneous readouts, and sophisticated pooled screening technology that takes advantage of powerful biological selection in vivo -- to extend our understanding of immune function in diverse cellular contexts. PUBLIC HEALTH RELEVANCE: Although all human genes have been identified, the actual functions of most genes are still mysterious. Recently, an experimental technique known as RNAi screening has become very useful to understand how genes function. We are proposing to set up a facility for RNAi screening that would allow scientists to ask fundamental questions relating to how the body fights off infectious and autoimmune diseases and cancers. This important resource would be broadly available to the general scientific community.