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 Core D 是该 U19 计划的核心任务，因为它将执行 CRISPR/Cas9 在小鼠中进行基因筛选，使 U19 研究人员能够识别新的调控因子 小鼠模型中对急性感染的先天和适应性免疫反应。核心 D 将制作一个 所有人都可以轻松进行 Cas9/CRISPR 介导的遗传筛选的创新技术 U19调查员。 Core D将协助U19调查人员进行屏幕设计；生成并验证骨骼 携带精心设计的 sgRNA 池的骨髓嵌合小鼠；感染这些骨髓嵌合体或 携带来自这些带有 U19 病毒的骨髓嵌合体的 sgRNA 的 T 细胞的接受者 调查员；并协助 U19 研究人员验证急性病毒感染的候选监管者 模型。核心 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 体内进行正向遗传筛选的方法。为了实现这些目标，具体目标 核心 D 是：1) 生成并验证携带 sgRNA 文库的骨髓嵌合小鼠。核心D 将提供屏幕设计建议，然后转出合适的Cas9表达 造血祖细胞带有慢病毒，携带由 Core C 生成的精选 sgRNA 文库； 2)用病毒感染骨髓嵌合小鼠或这些小鼠的T细胞受体。核心D将 感染骨髓嵌合体或来自骨髓嵌合体的转导T细胞的接受者用于体内 筛选（使用 LCMV Armstrong），以及先天性候选调节剂的体内验证研究 以及对急性感染的适应性免疫反应（使用 LCMV 阿姆斯特朗或流感病毒）。核心D 还将提供来自骨髓嵌合体的转导 DC，用于离体筛选以鉴定调节因子 DC与病原体、病原体成分​​和T细胞的相互作用； 3) 生成、维护和 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。 Core D 将作为表达 Cas9 的小鼠品系的储存库 病毒库存。这种集中的方法将使执行所需的一套程序标准化。 体内正向遗传筛选。通过核心提供这项新技术提供了一种高效且 以具有成本效益的方式进行这些体内筛选，以确保使用这些方法的一致性和成功 U19 研究人员的方法，并促进整个 U19 计划数据的比较。核 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和 验证候选调节因子，并使用 Core C 进行 sgRNA 慢病毒池的筛选，使用 Core A 进行筛选 通过以下方式促进核心 D 活动和核心 B 的沟通和行政监督 项目研究人员对免疫群体的单细胞数据进行计算分析。