NHGRI/DIR Embryonic Stem Cell and Transgenic Mouse Core

NHGRI/DIR 胚胎干细胞和转基因小鼠核心

• 批准号: 10267134
• 负责人: Lisa Garrett
• 金额: $ 144.7万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10267134
• 关键词: Alleles; Animal Model; Animals; Archives; Area; Bacteria; Breeding; Cell Line; Cells; Chimera organism; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Cryopreservation; DNA; Disasters; Dissection; ES Cell Line; Electroporation; Embryo; Female; Fertilization in Vitro; Fibroblasts; Freezing; Gene Targeting; Gene Transfer Techniques; Gene-Modified; Generations; Genes; Genetically Engineered Mouse; Genotype; Goals; Guide RNA; Harvest; Hybrid Cells; Hybrids; In Vitro; Injections; Knockout Mice; Location; MAP Kinase Gene; Methods; Modeling; Mus; Mutant Strains Mice; National Human Genome Research Institute; Natural Immunity; Pathway interactions; Production; Protocols documentation; Reagent; Research; Research Personnel; Ribonucleases; Services; Site; Standardization; System; Techniques; Technology; Teratoma; Time; Transfection; Transgenic Mice; Transgenic Organisms; Work; design and construction; embryo cell; embryonic stem cell; experimental study; genome editing; immunocytochemistry; in vivo; induced pluripotent stem cell; inhibitor/antagonist; male; mutant mouse model; nuclease; pluripotency; programs; sperm cell; stem cell technology; user-friendly; zygote

The NHGRI Embryonic Stem Cell and Transgenic Mouse Core provides a shared service to NHGRI investigators. The Core specializes in generating genetically engineered mice (GEM) via conventional transgenesis, ES cell targeting, but primarily through genome editing using site-specific nucleases (CRISPR/Cas). To do this work, the Core breeds, in-house, 90% of the animals to be used for generating GEM. The Core utilizes several background strains for generating mice and has developed and characterized embryonic stem cells from C57Bl/6J, C57Bl/6N, C57Bl/6 albino, 129S6Sv/Ev, and 129.B6(GFP). We also have a colony of various Cre expressing mice and other transgenics that are used by investigators across many protocols, thereby maximizing the efficiency and reducing mouse numbers by breeding for multiple users. All important mice are cryopreserved and stored in 2 separate locations for disaster purposes. All imported mice are rederived into the Core by in vitro fertilization. We routinely genotype mice and have adapted efficient protocols to minimize mice, reagents and time. The Core supports NHGRI investigators in construct design for gene targeting/editing and transgenesis, and in basic manipulations of the mouse and husbandry. Other services provided by the Core to help serve NHGRI investigators include teratoma analysis, embryo harvest and dissection, isolation of mouse embryonic fibroblasts and generation of induced pluripotent stem cells. These services allow NHGRI investigators to take full advantage of the mouse as a model organism. Thus, the Core not only generates mice, but we facilitate and enable investigators to perform mouse experiments in areas where they have less expertise. The NHGRI Transgenic Core is at the forefront of a major increase in transgenic production using CRISPR/Cas gene-editing constructs via either injection or electroporation of fertilized eggs. More recently, the core is developing protocols for in vivo gene editing or iGONAD. These technologies are replacing the generation of conventional knockout mice using embryonic stem cells and has made the technology more user friendly to a variety of investigators and allows NHGRI to be at the cutting edge of this new and important genomic editing technology in mammalian models. The core has generated targeted transgenics using the CRISPR/Cas system for hundreds of constructs since the technology was first introduced in the mouse( October 2013). Additionally, more transgenic/ gene edited lines have been cryopreserved due to the efficiency of CRISPR/Cas to generate multiple alleles.

NHGRI胚胎干细胞和转基因小鼠核心为NHGRI研究人员提供共享服务。核心专门通过传统的转基因，ES细胞靶向，但主要是通过使用位点特异性核酸酶（CRISPR/Cas）的基因组编辑来产生基因工程小鼠（GEM）。为了完成这项工作，Core内部繁殖了90%的动物，用于生产GEM。核心利用几种背景品系来产生小鼠，并且已经开发和表征了来自C57 Bl/6J、C57 Bl/6N、C57 Bl/6白化病、129 S6 Sv/Ev和129. B6（GFP）的胚胎干细胞。我们还有一个由各种Cre表达小鼠和其他转基因小鼠组成的群体，研究人员在许多方案中使用这些小鼠和转基因小鼠，从而通过为多个用户繁殖来最大限度地提高效率并减少小鼠数量。所有重要的小鼠都被冷冻保存，并储存在2个不同的地点，以备灾难之需。所有进口小鼠均通过体外受精再衍生为核心。我们常规地对小鼠进行基因分型，并采用有效的方案来最大限度地减少小鼠、试剂和时间。核心支持NHGRI研究人员在基因靶向/编辑和转基因的构建设计，并在小鼠和饲养的基本操作。核心提供的其他服务，以帮助服务NHGRI研究人员包括畸胎瘤分析，胚胎收获和解剖，小鼠胚胎成纤维细胞的分离和诱导多能干细胞的产生。这些服务使NHGRI研究人员能够充分利用小鼠作为模式生物。因此，核心不仅产生小鼠，而且我们促进并使研究人员能够在他们缺乏专业知识的领域进行小鼠实验。 NHGRI转基因核心站在使用CRISPR/Cas基因编辑构建体通过注射或电穿孔受精卵进行转基因生产的最前沿。最近，核心是开发体内基因编辑或iGONAD的协议。这些技术正在取代使用胚胎干细胞的传统基因敲除小鼠的产生，并使该技术对各种研究人员更加用户友好，并使NHGRI成为哺乳动物模型中这种新的重要基因组编辑技术的最前沿。自从该技术首次引入小鼠（2013年10月）以来，该核心已经使用CRISPR/Cas系统为数百个构建体产生了靶向转基因。此外，由于CRISPR/Cas产生多个等位基因的效率，更多的转基因/基因编辑品系已经被冷冻保存。