NHGRI/DIR Embryonic Stem Cell and Transgenic Mouse Core

NHGRI/DIR 胚胎干细胞和转基因小鼠核心

基本信息

批准号：
10691160
负责人：
Lisa Garrett
金额：
$ 158.17万
依托单位：
NATIONAL HUMAN GENOME RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/10691160
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 male mutant mouse model nuclease pluripotency programs sperm cell stem cell technology user-friendly zygote

项目摘要

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 has developed in house protocols for in vivo gene-editing and editing by electroporation with IVF generated embryos. 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研究者提供了共享服务。核心专门通过常规转基因（ES细胞靶向）生成基因工程小鼠（GEM），但主要是通过使用位点特异性核酸酶（CRISPR/CAS）进行基因组编辑。为了完成这项工作，核心在内部繁殖了90％用于生成宝石的动物。核心利用几种背景菌株生成小鼠，并从C57BL/6J，C57BL/6N，C57BL/6 Albino，129S6SV/EV和129.B6（GFP）开发并表征了胚胎干细胞。我们还拥有各种表达小鼠和其他转基因的殖民地，这些殖民地在许多方案中使用，这些殖民地通过许多用户繁殖，从而最大程度地提高了效率和减少鼠标数量。所有重要的小鼠都是冷冻保存的，并存储在两个单独的位置，用于灾难目的。所有进口小鼠都通过体外受精重新修复为核心。我们定期进行基因型小鼠，并具有适应有效的方案，以最大程度地减少小鼠，试剂和时间。核心支持NHGRI研究人员的构造设计，用于基因靶向/编辑和转基因以及小鼠和饲养的基本操作。核心提供的其他服务为NHGRI研究者提供服务，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以及诱导的多能干细胞的产生。这些服务使NHGRI调查人员可以充分利用鼠标作为模型生物。因此，核心不仅会产生小鼠，而且我们促进并使研究人员能够在专业知识较少的领域进行鼠标实验。 NHGRI转基因核心是通过CRISPR/CAS基因基因编辑构建体通过注射或施肥卵电穿孔来重大增加转基因的最前沿。最近，该核心已在用于体内基因编辑和通过IVF产生的胚胎的室内编辑和编辑的内部协议中开发。这些技术正在使用胚胎干细胞代替常规敲除小鼠的产生，并使该技术对各种研究人员更加用户友好，并允许NHGRI处于哺乳动物模型中这种新颖重要的基因组编辑技术的最前沿。自从该技术首次引入鼠标（2013年10月）以来，核心已使用CRISPR/CAS系统为数百种结构生成了有针对性的转基因。此外，由于CRISPR/ CAS产生多个等位基因的效率，因此更加转基因/基因编辑的线被冷冻保存。