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是这个U19程序的核心任务，因为它将执行 CRISPR/Cas9在小鼠中进行正向基因筛查，使U19研究人员能够识别新的调控因子 在小鼠模型中对急性感染的先天和获得性免疫反应。核心D将使一个 进行Cas9/CRISPR介导的基因筛查的创新技术，所有人都可以轻松获得 U19调查人员。核心D将协助U19调查人员进行屏幕设计；生成并验证骨骼 携带有精选sgRNA池的骨髓嵌合小鼠；感染这些骨髓嵌合体或 携带有U19病毒的骨髓嵌合体sgRNA的T细胞的接受者 调查人员；并协助U19调查人员验证急性病毒感染的候选调节剂 模特们。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 在活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)制备和验证携带sgRNA文库的骨髓嵌合小鼠。核心D 我将提供关于屏幕设计的建议，然后转换适当的Cas9-表达 携带慢病毒的造血祖细胞携带由核心C产生的sgRNAs的精选文库； 2)用病毒感染骨髓嵌合小鼠或来自这些小鼠的T细胞受体。核心D将 体内感染骨髓嵌合体或来自骨髓嵌合体的转导T细胞的受体 筛选(使用LCMV Armstrong)，并用于先天候选调节子的体内验证研究 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树突状细胞，用于体外筛查以识别调节器 DC与病原体、病原体成分和T细胞的相互作用；3)产生、维持和 验证TCR转基因和免疫谱系特异性表达Cas9的小鼠以及LCMV Armstrong 和流感病毒库存。核心D将作为表达Cas9的小鼠品系和 病毒式股票。这种集中的方法将标准化所需的一套程序 在活体内进行基因筛选。由核心提供的这种新技术提供了高效和 以经济高效的方式进行这些活体筛查，以确保使用这些筛查的一致性和成功 U19调查人员的方法，并促进整个U19项目的数据比较。堆芯 D将与项目1和2的调查人员密切合作，帮助他们进行活体筛查和 验证候选调节器，并使用核心C用于筛选sgRNA慢病毒池，核心A至 促进核心D和核心B活动的沟通和行政监督 来自免疫群体的单细胞数据的计算分析的项目调查人员。