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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：
9570589
负责人：
Lisa Garrett
金额：
$ 133.58万
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/9570589
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 Embryo Ethylnitrosourea 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 Injection of therapeutic agent Knockout Mice Location MAP Kinase Gene Methods Modeling Mus Mutant Strains Mice National Human Genome Research Institute Natural Immunity Oocytes Pathway interactions Production Protocols documentation Reagent Research Research Personnel Ribonucleases Services Site Standardization System Technology Teratoma Time Transfection Transgenic Mice Transgenic Organisms Work design and construction embryo cell embryonic stem cell experimental study genome editing immunocytochemistry induced pluripotent stem cell inhibitor/antagonist male mutant mouse model nuclease pluripotency programs sperm cell 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, and more recently 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 and transgenesis, and in basic manipulations of mouse husbandry. Other services provided by the Core to help serve NHGRI investigators include ENU injection, 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 injection into oocytes. This technology is 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 generated targeted transgenics using the CRISPR/Cas system for over 100 constructs since January 2014. Additionally, there were over 100 transgenic lines 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研究人员提供了共享服务。Core专门通过传统的转基因、ES细胞靶向，以及最近使用位点特异性核酸酶(CRISPR/CAS)的基因组编辑来生成基因工程小鼠(GEM)。为了完成这项工作，Core在内部饲养了90%用于制造宝石的动物。Core利用了几种背景菌株来产生小鼠，并开发了C57BL/6J、C57BL/6N、C57BL/6白化、129S6Sv/EV和129.B6(GFP)的胚胎干细胞并进行了鉴定。我们还有一群表达Cre的小鼠和其他转基因动物，研究人员可以跨多种方案使用它们，从而通过为多个用户繁殖来最大化效率并减少小鼠数量。所有重要的小鼠都被冷冻保存，并存放在两个不同的位置，以备灾难之用。所有进口的小鼠都通过体外受精重新获得核心。我们常规地对小鼠进行基因分型，并采用了有效的方案，以最大限度地减少小鼠、试剂和时间。该核心支持NHGRI研究人员进行基因打靶和转基因的构建设计，以及小鼠饲养的基本操作。Core为NHGRI研究人员提供的其他服务包括ENU注射、畸胎瘤分析、胚胎采集和解剖、小鼠胚胎成纤维细胞的分离以及诱导多能干细胞的产生。这些服务使NHGRI的研究人员能够充分利用小鼠作为模式生物的优势。因此，核心不仅产生老鼠，而且我们促进并使研究人员能够在他们专业知识较少的领域进行老鼠实验。 NHGRI转基因核心是通过注射到卵母细胞中使用CRISPR/Cas基因编辑构建物来大幅增加转基因生产的前沿。这项技术正在取代使用胚胎干细胞产生的传统基因敲除小鼠，并使该技术对各种研究人员更加用户友好，并使NHGRI处于这一新的、重要的哺乳动物模型基因组编辑技术的前沿。自2014年1月以来，核心利用CRISPR/CAS系统为100多个结构产生了有针对性的转基因。此外，由于CRISPR/CAs具有产生多个等位基因的效率，目前已有100多个转基因品系超低温保存。