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: 10901696
• 负责人: Lisa Garrett
• 金额: $ 191.56万
• 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/10901696
• 关键词: Alleles; 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 Modified; Gene Targeting; Gene Transfer Techniques; 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; Services; Site; Standardization; System; Techniques; Technology; Teratoma; Time; Transfection; Transgenic Mice; Transgenic Organisms; Work; blastomere structure; design and construction; embryo cell; embryonic stem cell; experimental study; genome editing; immunocytochemistry; in vivo; induced pluripotent stem cell; inhibitor; male; model organism; 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 embryonic 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，但主要是通过使用位点特异性核酸酶(CRISPR/CAS)进行基因组编辑来产生基因工程小鼠(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/编辑和转基因的构造设计，以及对老鼠和畜牧业的基本操作。Core为NHGRI研究人员提供的其他服务包括畸胎瘤分析、胚胎采集和解剖、小鼠胚胎成纤维细胞的分离和胚胎干细胞的生成。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转基因核心处于通过注射或电穿孔受精卵使用CRISPR/Cas基因编辑结构的转基因生产的主要增长的前沿。最近，该核心已经开发出内部协议，用于体内基因编辑和通过试管受精产生的胚胎进行电穿孔编辑。这些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。自从该技术首次引入小鼠(2013年10月)以来，核心已经使用CRISPR/CAS系统为数百个结构产生了有针对性的转基因。此外，由于CRISPR/CAs能产生多个等位基因，更多的转基因/基因编辑品系已被超低温保存。

项目成果

Method of euthanasia influences the oocyte fertilization rate with fresh mouse sperm.

安乐死方法影响新鲜小鼠精子的卵母细胞受精率。

• 发表时间: 2014
• 期刊: Journal of the American Association for Laboratory Animal Science : JAALAS
• 作者: Hazzard,KarenC;Watkins-Chow,DawnE;Garrett,LisaJ
• 通讯作者: Garrett,LisaJ

Author Correction: Mutations in COMP cause familial carpal tunnel syndrome.

作者更正：COMP 突变导致家族性腕管综合征。

• DOI: 10.1038/s41467-020-17845-7
• 发表时间: 2020
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Li,Chunyu;Wang,Ni;Schäffer,AlejandroA;Liu,Xilin;Zhao,Zhuo;Elliott,Gene;Garrett,Lisa;Choi,NgaTing;Wang,Yueshu;Wang,Yufa;Wang,Cheng;Wang,Jin;Chan,Danny;Su,Peiqiang;Cui,Shusen;Yang,Yingzi;Gao,Bo
• 通讯作者: Gao,Bo