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

描述(申请人提供)：尽管基因组计划成功地列出了每一个基因，但关于大多数基因如何在复杂的生物过程中发挥作用，仍有许多需要了解。在哺乳动物中尤其如此，因为在哺乳动物中，高通量的遗传方法要么不可行，要么非常耗时。细胞系中的RNA干扰(RNAi)筛选已被证明在这方面非常有用；特别是利用体内生物选择的力量的RNAi和微型RNA(MiRNA)筛选在未来具有巨大的潜力。在本申请中，我们申请资金来建立一个RNAi/miRNA筛选设施，以解决RFA的五个研究主题中的两个。(I)该设施将通过提供下文概述的大规模RNAi和miRNA筛选所需的高通量仪器和RNAi/miRNA库，极大地增强我们将基因组学和其他高通量技术应用于免疫功能研究的能力。(Ii)作为一种在拟议的3年项目期结束时能够自给自足的资源，该设施将通过提供有关RNAi屏幕设计的仪器、试剂和建议，重振圣地亚哥的当地生物医学研究界，我们相信这些仪器、试剂和建议将促进新的发现、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问题。在项目1(LIAI)中，我们将使用全基因组siRNA筛选来确定细胞对双链(Ds)RNA反应中缺失的角色，并使用聚焦慢病毒shRNA筛选来确定启动对外来微生物dsDNA的反应的未知DNA结合蛋白(S)。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，以了解它们在体内消除B细胞耐受的能力。在项目3(LIAI)中，我们将使用该miRNA文库，以及针对转录因子和染色质相关蛋白的聚焦慢病毒shRNA文库，以确定影响CD8T细胞表达效应标记或记忆细胞命运标记的候选基因。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(干扰素-3)能力的转录因子和染色质相关蛋白，首先是在培养的人类细胞系中，然后是在体内汇集的shRNA屏幕中。总之，这些研究使用最先进的方法--基于细胞的筛查格式，具有多个同时读数，以及利用体内强大的生物选择的复杂的汇集筛查技术--来扩展我们对不同细胞环境中免疫功能的理解。 与公共卫生相关：尽管所有人类基因都已被确认，但大多数基因的实际功能仍然是个谜。最近，一种名为RNAi筛选的实验技术对于了解基因的功能变得非常有用。我们提议建立一个RNAi筛查设施，允许科学家提出与人体如何抵御传染病和自身免疫性疾病以及癌症有关的基本问题。这一重要资源将广泛提供给一般科学界。

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.