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

作为对NHGRI研究者的服务，胚胎干细胞和转基因小鼠核心专门针对基因功能和调节的基本研究以及创建人类遗传疾病的小鼠模型的基本研究。核心利用了几种技术来产生遗传改变的小鼠。第一种方法是通过将DNA显微注射到受精的胚胎中（核微注射）来产生常规转基因，以产生生成小鼠。其次，靶向转基因是通过显微注射遗传改变的胚胎干细胞（ES细胞）产生的。通过从其他机构（即IKMC）导入的ES细胞系的靶向基因的同源重组（IKMC）导入的线需要扩展和归档，DNA/RNA分析，Karyotyping和MAP/MAP/MAPLASMA测试。然后将ES细胞注入2.5天的8细胞胚胎或3.5天的胚泡，分别产生完全ES细胞衍生或嵌合小鼠。核心还通过与杂化ES细胞的四倍体胚胎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产生杂合或ES细胞衍生的小鼠，繁殖嵌合体所需的动物时间和数量减少。最近，核心已经实施了使用位点特异性核酸酶（CRISPR/CAS和TALES）的使用，以一步一步产生突变小鼠，直接将DNA和RNA靶向注射到Egg pronuclei中。这种直接的成分可以生成具有多种基因修饰的生殖系小鼠。 核心存档，在多个位置，通过对精子和胚胎的冷冻保存，并通过体外受精来重构线条。因此，在发生灾难或爆发的情况下，核心可以迅速重新建立小鼠菌株，并更有效地向其他机构出口线。为了进行质量控制，核心冷冻水果来自野生型菌株（C57BL/6J，FVB/N，129S6SV/EV和BALB/C）的库存胚胎。这些胚胎还用于柔韧的8细胞胚胎和受精卵的微分注射。使用冷冻保存的胚胎可将动物供体的需求减少50-60％，并允许每次疗程多2-3倍胚胎。提供给我们研究所的另一项服务是通过胚胎转移受精卵的胚胎转移到我们的设施中。受精卵是由野生卵母细胞进口的雄性精子的IVF产生的。任何其他鸡蛋/精子都是冷冻保存的灾难。与常规隔离方法相比，重新启动通常需要7-8周，而14-16周。 与我们的重新培养和冷冻保存计划密切相关，是内部育种殖民地。该菌落迅速生成用于实验的小鼠，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）上繁殖，并冷冻保存线。我们可以很容易地重新建立小鼠，从而减少了架子空间以维护繁殖。其他服务包括胚胎解剖，小鼠灌注，注射，菌落维持和通过基因分型鉴定。该核心与NHGRI调查人员在构造设计和小鼠饲养的基本操作中合作。 核心致力于尖端的转基因技术，同时找到更好的途径来减少动物需求。例如，我们正在研究抑制剂，以改善ES细胞质量和改善的嵌合体。通过使用GSK3B和MAPK的抑制剂，我们的父母细胞系和目标线具有更好的形态，受控的生长速率和较高的纳米水平。这应该改善内部和多个导入的ES细胞。 核心已成功生成的MIP衍生出已针对的干细胞，并将通过细菌传播。我们为MEF或尾尖成纤维细胞准备，并将它们提供给我们的调查人员，他们要求它们进行人类ES和IPS研究。此外，我们通过协助裸鼠的细胞注射和产生细胞的细胞注射和产生来支持人类IPS线的表征。 核心是使用TALES和CRISPR/CAS系统的基因组编辑方案。位点特定的核酸酶系统允许在一个步骤过程中生产具有多个基因修饰的基因工程小鼠。 核心人员描述和设备功能： 转基因核心有七个全职员工。丽莎·加勒特（Lisa Garrett）指导并监督日常操作，培训和实验设计。核心人员包括六名员工，直接通过核心，通过分支机构或合同雇用。其中包括Jun Cheng，Gene Elliott，Kowser Haseen，Karen Hazzard（所有技术人员）以及Cecilia Rivas和Elsa Escobar（动物支持承包商）。 核心的物理组织分为动物屏障后面的两个实验室。三楼实验室设有ES细胞生长和维护的中央组织培养空间，一个冷冻保存区域，一个带有三个Zeiss/Eppendorf微注射站的微注射套件和一个Nikon微观启发站，一个分子生物学区域以及5个人的行政空间。二楼的实验室用于动物使用，配以向下气流桌和生物安全柜。我们有一个专门的区域，用于收集4个立体显微镜的胚胎和组织，可用于研究人员。该实验室包含一个用于ENU的化学通风罩，一个带有3个立体显微镜和下降桌子的小型动物手术套件，用于IPS生成的组织培养区域以及ES细胞分化。 有两个人的行政空间。 摘要2013-2014 自2013年7月以来，核心利用了42个DNA构建体（包括CRISPR/CAS和TALEN）的常规转基因。我们有29个ES细胞靶向构造，这些构建体处于各个开发阶段，例如筛选出同源重组，微注射，并产生种系的产生后代。我预计即将到来的一年中将有20多种传统的转基因结构，并至少为我们的研究所提供35个目标结构。 在过去的一年中，我们在进口小鼠的15行，冷冻保存的31行进行灾难准备和归档的57条鼠标线上重新修复。在接下来的一年中，我们将继续冷冻保存所有由核心进口或产生的突变小鼠。我已经修改了我们的冷冻程序，以减少冷冻的胚胎数量（因此减少了小鼠的数量），并用冷冻保存精子来平衡存档。我们使用Nakagata等人的修改方法，J.Mamm.ova Res恢复了IVF（> 50％）回收冷冻精子的效率显着提高。 2010。 除了我们的C57BL6/J和Hybrid 129.b6细胞系，核心在过去一年中还产生了三种新的胚胎干细胞系。现在，我们拥有竞争性的129S6和B6/J白化胚胎干细胞。此外，我们最近生成了C57BL6/NES细胞，并正在测试嵌合和种系传播。 从审查PubMed和我们的研究人员的信息，该核心在2005年至今的120篇论文中做出了重大贡献。这包括核心几个成员的共同作者和致谢。论文的汇编表示由核心生成的任何资源或鼠标。自2012年1月以来，核心已经在至少4个出版物上共同撰写