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: 8948412
• 负责人: Lisa Garrett
• 金额: $ 139.86万
• 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/8948412
• 关键词: Animals; Archives; Area; Authorship; Breeding; Cell Line; Cell Maintenance; Cells; Chemicals; Chimera organism; Chimerism; Clustered Regularly Interspaced Short Palindromic Repeats; Commit; Contractor; Contracts; Cryopreservation; DNA; Development; Disasters; Disease Outbreaks; Dissection; ES Cell Line; Embryo; Embryo Transfer; Employee; Equilibrium; Equipment; Ethylnitrosourea; Event; Experimental Designs; Female; Fertilization in Vitro; Fibroblasts; Floor; Freezing; Gene Expression Regulation; Gene Targeting; Gene-Modified; Generations; Genes; Genetically Engineered Mouse; Genome; Genotype; Germ Lines; Goals; Growth; Harvest; Hereditary Disease; Housing; Human; Human Genetics; Human Resources; Hybrids; In Vitro; Individual; Injection of therapeutic agent; Institutes; Institution; Karyotype determination procedure; Laboratories; Location; MAP Kinase Gene; Maintenance; Methods; Microinjections; Micromanipulation; Molecular Biology; Morphology; Mouse Strains; Mus; Mutant Strains Mice; Mycoplasma; National Human Genome Research Institute; Nude Mice; One-Step dentin bonding system; Oocytes; Operative Surgical Procedures; Ovum; Paper; Pathway interactions; Perfusion; Preparation; Process; Production; Protocols documentation; PubMed; Publications; Quality Control; Quarantine; RNA; RNA analysis; Research; Research Personnel; Resources; Services; Site; Space Maintenance; Staging; Stem cells; System; Tail; Technology; Teratoma; Testing; Time; Training; Transgenic Mice; Transgenic Organisms; Work; base; blastocyst; design and construction; egg; embryo cell; embryo tissue; embryonic stem cell; flexibility; gene function; homologous recombination; immunocytochemistry; improved; induced pluripotent stem cell; inhibitor/antagonist; male; member; mouse model; mutant; mutant mouse model; nuclease; operation; pluripotency; programs; reconstitution; research study; screening; sperm cell; stem cell differentiation; tissue culture; transmission process; zygote

As a service to NHGRI investigators, the Embryonic Stem Cell and Transgenic Mouse Core specializes in generating genetically altered mouse lines for basic studies of gene function and regulation and for the creation of mouse models of human genetic diseases. Several technologies are utilized by the Core to generate genetically altered mice. The first method is to create conventional transgenics by microinjection of DNA into fertilized embryos (pronuclear microinjection) to generate germline mice. Secondly, targeted transgenics are generated by microinjecting genetically altered embryonic stem cells (ES cells). The ES cells are modified via homologous recombination of targeted genes in the Core or, are imported ES cell lines from other institutions (i.e., IKMC) Imported lines require expansion and archiving, DNA/RNA analysis, karyotyping, and MAP/Mycoplasma testing. ES cells are then injected into 2.5 day 8-cell embryos or 3.5 day blastocysts to generate fully ES cell-derived or chimeric mice, respectively. The Core also generates transgenic mice by aggregation of tetraploid embryos with hybrid ES cells. By using 8-cell or tetraploid aggregation embryos to produce heterozygous or fully ES cell derived mice, the time and number of animals required is reduced for breeding chimeras. Recently, the Core has implemented the use of site-specific nucleases (CRISPR/Cas and TALENs)to generate mutant mice in one step with direct, targeted injection of DNA and RNA into egg pronuclei. This direct inception can generate germ line mice with multiple gene modifications. The Core archives, in multiple locations, mutant strains by cryopreservation of sperm and embryos and reconstitutes the lines by in vitro fertilization. Thus, the Core can rapidly re-establish mouse strains in the event of a disaster or outbreak as well as easily export lines more efficiently and humanely to other institutions. For quality control, the Core cryopreserves stock embryos from wild-type strains (C57Bl/6J, FVB/N, 129S6Sv/Ev and Balb/c). These embryos are also used for flexible microinjection of 8-cell embryos and fertilized eggs. The use of cryopreserved embryos reduces animal donor needs by 50-60% and allows microinjection of 2-3x more embryos per session. An additional service provided to our institute is to rederive animals into our facility by embryo transfer of fertilized eggs. Fertilized eggs are generated by IVF of imported male sperm with wildtypye oocytes. Any additional eggs/sperm are cryopreserved for disaster. Rederivation generally takes 7-8 weeks as compared to 14-16 weeks for conventional quarantine methods. Closely associated to our rederivation and cryopreservation program, is the in-house breeding colony. This colony rapidly generates mice for experiments and centralizes animals used across animal protocols (ie. cre transgenics). We perform PCR-based genotyping, breed on multiple backgrounds (129 and C57Bl/6) and cryopreserve the lines. We can readily reconstitute mice, which enables reduced rack space for maintenance breeding. Other services include embryo dissection, mouse perfusions, injections, colony maintenance, and animal identification by genotyping. The Core works with NHGRI investigators in construct design, and in basic manipulations of mouse husbandry. The Core is committed to cutting edge transgenic technologies while finding better avenues to reduce the animal requirements. For example, we are investigating inhibitors to improve ES cell quality and improved chimeras. By using inhibitors of GSK3b and MAPK, our parental cell lines and targeted lines have better morphology, controlled growth rate, and higher Nanog levels. This should improve both in-house, and the multiple imported ES cells. The Core has successfully generated mIPS derived stem cells that have been targeted and will transmit through the germ line. We prepare MEFS or tail tip fibroblasts and make them availablee to our investigators who require them for human ES and iPS studies. Moreover, we support characterization of human iPS lines by assisting in the cell injection and generation of teratomas in nude mice. The Core is utilizing protocols for genome editing using TALENs and Crispr/Cas systems. The site specific nuclease systems allows production of genetically engineered mice with multiple gene modifications in a one step process. Core Personnel Description and Equipment Capabilities: The Transgenic Core has seven full-time employees. Lisa Garrett directs and oversees daily operations, training and experimental design. The core staff includes six staff, employed directly by the core, through a branch, or by contract. They include Jun Cheng, Gene Elliott, Kowser Hasneen, Karen Hazzard (all technical staff) and Cecilia Rivas and Elsa Escobar (animal support contractors). The physical organization of the Core is divided into two laboratories behind the animal barrier. The third floor lab houses the central tissue culture space for ES cell growth and maintenance, a cryopreservation area, a microinjection suite with three Zeiss/Eppendorf microinjection stations and one Nikon micromanipulation station, a molecular biology area, and administrative space for 5 individuals. The second floor laboratory is for animal use with downdraft tables and biosafety cabinets. We have a dedicated area for harvesting embryos and tissues with 4 stereomicroscopes that are available to institute investigators. This lab contains a chemical fume hood for ENU, a small animal surgery suite with 3 stereomicroscopes and downdraft tables, a tissue culture area for iPS generation, and ES cell differentiation. There is administrative space for 2 individuals. Summary July 2013-2014 The Core utilized conventional transgenics from 42 DNA constructs( including CRISPR/Cas and TALEN) since July 2013. We have 29 ES cell - targeting constructs that are in various stages of development such as screening for homologous recombination, microinjection, and generation of germline transmitting progeny. I anticipate greater than 20 conventional transgenic constructs for the upcoming year and at least 35 targeting constructs for our institute. During the past year, we rederived in 15 lines of imported mice, cryopreserved 31 lines for disaster preparation, and archived 57 mouse lines. For the upcoming year, we will continue to cryopreserve all mutant mice imported or generated by the Core. I have modified our freezing program to reduce the numbers of embryos frozen (therefore reducing the numbers of mice) and balance the archiving with cryopreserved sperm. We have a significant increase in efficiency of recovering frozen sperm by IVF (>50%) using a modified method by Nakagata et al., J.Mamm.Ova Res. 2010. The Core has generated three new embryonic stem cell lines this past year in addition to our C57Bl6/J and hybrid 129.B6 cell lines. We now have germline competent 129S6 and B6/J albino embryonic stem cells. In addition, we have recently generated C57Bl6/N ES cells and are testing for chimerism and germline transmission. From reviewing Pubmed and information from our investigators, the Core has made substantial contributions on >120 papers from 2005-present. This includes both co-authorship and acknowledgements of several members of the Core. This compilation of papers represents any resource or mouse generated by the Core. Since January 2012, the Core has had co-authorship on at least 4 publications

作为为 NHGRI 研究人员提供的服务，胚胎干细胞和转基因小鼠核心专门生产转基因小鼠品系，用于基因功能和调控的基础研究以及创建人类遗传疾病的小鼠模型。 Core 使用多种技术来产生基因改造小鼠。第一种方法是通过将 DNA 显微注射到受精胚胎（原核显微注射）中来产生种系小鼠，从而产生常规转基因。其次，通过显微注射基因改变的胚胎干细胞（ES细胞）产生靶向转基因。 ES 细胞通过 Core 中目标基因的同源重组进行修饰，或者是从其他机构（即 IKMC）导入的 ES 细胞系。导入的细胞系需要扩展和存档、DNA/RNA 分析、核型分析和 MAP/支原体测试。然后将 ES 细胞注射到 2.5 天的 8 细胞胚胎或 3.5 天的囊胚中，分别产生完全 ES 细胞衍生的小鼠或嵌合小鼠。 Core 还通过四倍体胚胎与杂交 ES 细胞的聚集来产生转基因小鼠。通过使用8细胞或四倍体聚集胚胎来产生杂合或完全ES细胞衍生的小鼠，可以减少培育嵌合体所需的时间和动物数量。最近，Core 已经实现了使用位点特异性核酸酶（CRISPR/Cas 和 TALEN）将 DNA 和 RNA 直接定向注射到卵原核中，一步生成突变小鼠。这种直接起始可以产生具有多种基因修饰的种系小鼠。 核心档案在多个地点通过冷冻保存精子和胚胎产生突变株，并通过体外受精重建品系。因此，在发生灾难或疫情爆发时，核心可以快速重建小鼠品系，并且可以更有效、更人道地轻松地将品系出口到其他机构。为了进行质量控制，Core 冷冻保存野生型菌株（C57Bl/6J、FVB/N、129S6Sv/Ev 和 Balb/c）的储备胚胎。这些胚胎还用于 8 细胞胚胎和受精卵的灵活显微注射。使用冷冻胚胎可将动物捐赠需求减少 50-60%，并且每次可显微注射 2-3 倍的胚胎。向我们研究所提供的一项额外服务是通过受精卵胚胎移植将动物重新引入我们的设施。受精卵是通过进口男性精子与野生型卵母细胞的体外受精产生的。任何额外的卵子/精子都会被冷冻保存以备灾难之需。重新衍生通常需要 7-8 周，而传统检疫方法需要 14-16 周。 与我们的再衍生和冷冻保存计划密切相关的是内部繁殖群。该群体快速产生用于实验的小鼠，并集中在动物方案中使用的动物（即cre转基因）。我们进行基于 PCR 的基因分型，在多种背景（129 和 C57Bl/6）下进行繁殖并冷冻保存品系。我们可以轻松地重组小鼠，从而减少维持饲养的机架空间。其他服务包括胚胎解剖、小鼠灌注、注射、群体维持和通过基因分型进行动物鉴定。该核心与 NHGRI 研究人员合作进行构建设计和小鼠饲养的基本操作。 该核心致力于尖端转基因技术，同时寻找更好的途径来减少动物需求。例如，我们正在研究改善 ES 细胞质量和改进嵌合体的抑制剂。通过使用 GSK3b 和 MAPK 抑制剂，我们的亲本细胞系和靶向细胞系具有更好的形态、受控的生长速率和更高的 Nanog 水平。这应该会改善内部和多种进口的 ES 细胞。 Core 已成功生成 mIPS 衍生干细胞，这些干细胞已成为目标，并将通过生殖系传播。我们制备 MEFS 或尾尖成纤维细胞，并将其提供给需要它们进行人类 ES 和 iPS 研究的研究人员。此外，我们还通过协助裸鼠细胞注射和畸胎瘤生成来支持人类 iPS 系的表征。 Core 正在利用 TALEN 和 Crispr/Cas 系统进行基因组编辑协议。位点特异性核酸酶系统可以一步生产出具有多种基因修饰的基因工程小鼠。 核心人员描述及装备能力： Transgenic Core 拥有七名全职员工。丽莎·加勒特 (Lisa Garrett) 指导和监督日常运营、培训和实验设计。核心人员包括六名员工，由核心人员直接聘用、通过分支机构聘用或通过合同聘用。他们包括 Jun Cheng、Gene Elliott、Kowser Hasneen、Karen Hazzard（所有技术人员）以及 Cecilia Rivas 和 Elsa Escobar（动物支持承包商）。 核心的物理组织在动物屏障后面分为两个实验室。三楼实验室设有用于 ES 细胞生长和维持的中央组织培养空间、一个冷冻保存区、一个配备三个 Zeiss/Eppendorf 显微注射站和一个 Nikon 显微操作站的显微注射套件、一个分子生物学区域以及可容纳 5 人的行政空间。二楼实验室供动物使用，配有下吸式工作台和生物安全柜。我们有一个专门用于采集胚胎和组织的区域，配有 4 台立体显微镜，可供研究所研究人员使用。该实验室包含用于 ENU 的化学通风柜、配有 3 个立体显微镜和下吸式工作台的小动物手术套件、用于 iPS 生成和 ES 细胞分化的组织培养区。 有可容纳 2 人的行政空间。 2013年7月-2014年总结 自 2013 年 7 月以来，Core 使用了来自 42 个 DNA 构建体（包括 CRISPR/Cas 和 TALEN）的传统转基因技术。我们有 29 个 ES 细胞靶向构建体，它们处于不同的开发阶段，例如同源重组筛选、显微注射和生殖系传递后代的产生。我预计来年将有超过 20 个常规转基因构建体，我们研究所将至少有 35 个靶向构建体。 在过去的一年里，我们重新衍生了15个进口小鼠品系，冷冻保存了31个备灾品系，并归档了57个小鼠品系。来年，我们将继续冷冻保存由 Core 导入或产生的所有突变小鼠。我修改了我们的冷冻程序，以减少冷冻胚胎的数量（从而减少小鼠的数量）并平衡冷冻精子的存档。使用 Nakagata 等人 J.Mamm.Ova Res 的改进方法，我们通过 IVF 回收冷冻精子的效率显着提高 (>50%)。 2010年。 除了我们的 C57Bl6/J 和杂交 129.B6 细胞系外，Core 在去年还产生了三种新的胚胎干细胞系。我们现在拥有生殖系感受态 129S6 和 B6/J 白化胚胎干细胞。此外，我们最近生成了 C57Bl6/N ES 细胞，并正在测试嵌合和种系传递。 通过审查 Pubmed 和我们研究人员提供的信息，核心从 2005 年至今对超过 120 篇论文做出了重大贡献。这包括核心成员的共同作者和致谢。该论文汇编代表了核心生成的任何资源或鼠标。自 2012 年 1 月以来，Core 已共同撰写了至少 4 篇出版物