Core D: Mouse Perturbation Core

核心 D：鼠标扰动核心

• 批准号: 10207348
• 负责人: Arlene H. Sharpe
• 金额: $ 81.28万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-05 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207348
• 关键词: Acute; Bar Codes; Bioinformatics; Bone Marrow; Breeding; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cell Communication; Cells; Chimera organism; Communication; Computer Analysis; Custom; Data; Ensure; Gene Deletion; Genes; Genetic Screening; Genomic DNA; Goals; Hematopoietic stem cells; Human; Immune; Immunity; Infection; Influenza; Laboratories; Lentivirus; Libraries; Lymphocytic choriomeningitis virus; Mediating; Microbe; Mission; Modeling; Mouse Strains; Mus; Population; Procedures; Quality Control; Reagent; Reporter; Research Personnel; Screening Result; Screening procedure; Source; Standardization; System; T cell response; T-Lymphocyte; Technology; Transgenic Mice; Transgenic Organisms; Viral; Virus; Virus Diseases; Work; acute infection; adaptive immune response; candidate validation; cell type; congenic; cost effective; data management; design; experimental study; in vivo; influenzavirus; innovative technologies; interest; microbial; mouse model; new technology; novel; pathogen; programs; repository; single cell analysis; single-cell RNA sequencing; stem cells; success; validation studies

Mouse Perturbation Core D is central the mission of this U19 Program because it will execute CRISPR/Cas9 forward genetic screens in mice to enable U19 investigators to identify novel regulators of innate and adaptive immune responses to acute infection in mouse models. Core D will make an innovative technology for conducting Cas9/CRISPR-mediated genetic screens easily accessible to all U19 investigators. Core D will assist U19 investigators with screen design; generate and validate bone marrow chimeric mice carrying curated pools of sgRNA; infect these bone marrow chimeras or recipients of T cells carrying sgRNAs from these bone marrow chimeras with virus for U19 investigators; and assist U19 investigators with validation of candidate regulators in acute viral infection models. Core D has expertise with all of these procedures and demonstrated success with using these approaches to conduct forward genetic screens in vivo. To achieve these goals, the Specific Aims of Core D are: 1) To generate and validate bone marrow chimeric mice carrying sgRNA libraries. Core D will provide advice on screen design and then transduce the appropriate Cas9-expressing hematopoietic progenitor cells with lentivirus carrying curated libraries of sgRNAs generated by Core C; 2) To infect bone marrow chimeric mice or recipients of T cells from these mice with virus. Core D will infect bone marrow chimeras or recipients of transduced T cells from bone marrow chimeras for in vivo screens (using LCMV Armstrong), and for in vivo validation studies of candidate regulators of innate and adaptive immune responses to acute infection (using LCMV Armstrong or influenza virus). Core D also will provide transduced DCs from bone marrow chimeras for ex vivo screens to identify regulators of DC interactions with pathogens, pathogen components and T cells; 3) To generate, maintain and validate TCR transgenic and immune-lineage-specific Cas9 expressing mice, and LCMV Armstrong and influenza viral stocks. Core D will serve as a repository for Cas9-expressing mouse strains and viral stocks. This centralized approach will standardize the set of procedures needed to conduct forward genetic screens in vivo. Provision of this novel technology by a core provides an efficient and cost-effective means to conduct these in vivo screens, to ensure uniformity and success in use of these approaches by U19 investigators, and to facilitate comparisons of data across the U19 Program. Core D will work closely with investigators in Project 1 and 2 to help them conduct in vivo screens and validate candidate regulators, and with Core C for lentiviral pools of sgRNA for the screens, Core A to facilitate communication and administrative oversight of Core D activities, and Core B through the Project investigators for computational analyses of single cell data from immune populations.

鼠标扰动核心D是本U19计划的核心使命，因为它将执行 CRISPR/Cas9在小鼠中进行正向遗传筛选，使U19研究人员能够识别新的调节剂 小鼠模型中对急性感染的先天性和适应性免疫反应。核心D将使一个 用于进行Cas9/CRISPR介导的遗传筛选的创新技术，所有人都可以轻松获得 U19调查员核心D将协助U19研究者进行筛选设计;生成和确认骨 携带策划的sgRNA池的骨髓嵌合小鼠;感染这些骨髓嵌合体或 携带来自这些骨髓嵌合体的sgRNA的T细胞与U19病毒的受体 研究者;并协助U19研究者验证急性病毒感染中的候选调节剂 模型Core D拥有所有这些程序的专业知识，并在使用这些程序方面取得了成功 在体内进行正向遗传筛选的方法。为了实现这些目标， 核心D是：1）产生并验证携带sgRNA文库的骨髓嵌合小鼠。内核D 将提供有关筛选设计的建议，然后筛选适当的Cas9表达 具有慢病毒的造血祖细胞，所述慢病毒携带由核心C产生的sgRNA的策划文库; 2)用病毒感染骨髓嵌合小鼠或来自这些小鼠的T细胞的受体。核心D将 感染骨髓嵌合体或来自骨髓嵌合体的转导T细胞的受体体内形成 筛选（使用LCMV Armstrong），并用于先天免疫缺陷候选调节因子的体内验证研究。 和对急性感染的适应性免疫应答（使用LCMV Armstrong或流感病毒）。内核D 还将提供来自骨髓嵌合体的转导的DC用于离体筛选以鉴定调节因子 DC与病原体、病原体组分和T细胞相互作用的能力; 3）产生、维持和 验证TCR转基因和免疫谱系特异性Cas9表达小鼠，以及LCMV Armstrong 和流感病毒库存。核心D将作为表达Cas9的小鼠品系的储存库， 病毒性股票这种集中的办法将使开展工作所需的一套程序标准化， 在体内进行遗传筛选。通过芯提供这种新颖的技术提供了一种有效的且 进行这些体内筛选的成本有效的手段，以确保这些筛选的一致性和成功使用。 U19研究人员的方法，并促进整个U19计划的数据比较。核心 D将与项目1和2的研究人员密切合作，帮助他们进行体内筛选， 验证候选调节子，并与核心C用于筛选sgRNA的慢病毒库，核心A用于筛选sgRNA的慢病毒库，核心A用于筛选sgRNA的慢病毒库，核心C用于筛选sgRNA的慢病毒库，核心A用于筛选sgRNA的慢病毒库。 促进核心D活动和核心B活动的沟通和行政监督， 免疫群体单细胞数据计算分析项目研究者。