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

免疫功能的新参与者：通过 RNAi 和 micro-RNA 筛选进行识别

• 批准号: 8048192
• 负责人: MITCHELL KRONENBERG
• 金额: $ 1257.91万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2013-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8048192
• 关键词: Address; Arts; Autoimmune Diseases; B-Lymphocytes; Biological; Biological Process; Biomedical Research; Bone Marrow; CD8B1 gene; Cell Culture Techniques; Cell Line; Cell Nucleus; Cell Separation; Cell physiology; Cells; Chromatin; Collaborations; Communities; Competence; Complex; Cultured Cells; Cytokine Gene; Cytoplasm; DNA-Binding Proteins; Detection; Development; Double-Stranded RNA; Embryo; Endoplasmic Reticulum; Event; Fibroblasts; Funding; Future; Genes; Genetic; Genetic Transcription; Genome; Genomics; Grant; Human; Human Cell Line; Hypersensitivity; IRF3 gene; Immune Tolerance; Immune system; Immunity; Immunology; Individual; Institutes; Institution; Interferons; Interleukin 7 Receptor; Interleukin-4; Lead; Libraries; Malignant Neoplasms; Mammals; Memory; Messenger RNA; Methods; MicroRNAs; Mitochondria; Molecular; Molecular Immunology; Monitor; Mus; Nucleic Acid Binding; Pathway interactions; Process; Production; Proteins; Proteomics; RNA Interference; RNA library; Reagent; Reporter; Request for Applications; Research; Research Institute; Research Personnel; Resistance; Resources; Scientist; Screening procedure; Self Tolerance; Signal Transduction; Small Interfering RNA; Subfamily lentivirinae; T-Lymphocyte; Techniques; Technology; Time; Transcriptional Regulation; Transfection; Transgenic Mice; United States National Institutes of Health; Viral; antimicrobial; arm; base; crosslink; cytokine; design; fighting; functional genomics; gene function; genome-wide; high throughput technology; immune function; in vivo; instrument; instrumentation; microbial; pathogen; perforin; porin; precursor cell; protein function; public health relevance; reconstitution; response; sensor; skills; small hairpin RNA; success; transcription factor

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.

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，但关于大多数基因在复杂的生物过程中如何发挥作用，仍然有很多东西需要了解。在哺乳动物中尤其如此，因为高通量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，利用体内生物选择能力的RNAi和micro-RNA（miRNA）筛选在未来具有巨大的潜力。在这项申请中，我们要求资助建立一个RNAi/ miRNA筛选设施，解决RFA五个研究主题中的两个。(i)该设施将大大提高我们的能力，“应用基因组学和其他高通量技术”的免疫功能的研究，通过提供高通量仪器和RNAi/ miRNA文库所需的大规模RNAi和miRNA屏幕概述如下。(ii)作为一种资源，旨在在拟议的3年项目期结束时自我维持，该设施将通过提供有关RNAi屏幕设计的仪器，试剂和建议，“重振和激励圣地亚哥当地的生物医学研究社区”，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）的研究人员使用，以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筛选来识别对双链（ds）RNA的细胞应答中缺失的参与者，并使用聚焦慢病毒shRNA筛选来识别启动对外源微生物dsDNA应答的未知DNA结合蛋白。在项目2（TSRI）中，我们将筛选一个全面的表达miRNA的慢病毒库，以了解它们在体内消除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，以及针对转录因子和染色质相关蛋白的聚焦慢病毒shRNA文库，来鉴定影响CD 8 T细胞表达效应细胞或记忆细胞命运标志物的候选物。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细胞介素-4（IL-4）和干扰素-3（IFN-3）的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，然后在体内进行混合shRNA筛选。这些研究共同使用了最先进的方法-基于细胞的筛选格式，具有多个同时读出，以及利用体内强大的生物选择的复杂的合并筛选技术-以扩展我们对不同细胞背景下免疫功能的理解。 尽管人类所有的基因都已被鉴定，但大多数基因的实际功能仍然是个谜。最近，一种被称为RNAi筛选的实验技术对于了解基因的功能非常有用。我们建议建立一个RNAi筛选设施，使科学家能够提出与身体如何对抗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。