Genome Modification Core

基因组修饰核心

• 批准号: 7673007
• 负责人: JOSHUA R SANES
• 金额: $ 17.91万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7673007
• 关键词: Animal Husbandry; Animal Technicians; Animals; Arts; Biological; Biomedical Research; Biopsy; Breeding; Categories; Computers; Consultations; Core Facility; Cryopreservation; DNA; Depth; Derivation procedure; Development; Doctor of Philosophy; Educational process of instructing; Electroporation; Embryo; Embryo Transfer; Equipment; Equipment and Supplies; Faculty; Fertilization in Vitro; Floor; Funding; Gene Targeting; Generations; Genetic; Genetic Models; Genome; Grant; Green Fluorescent Proteins; Housing; Incubators; Individual; Injection of therapeutic agent; Institutes; Intention; Interneurons; Intracytoplasmic Sperm Injections; Knock-in Mouse; Knock-out; Laboratories; Lentivirus Vector; Liquid substance; Mental disorders; Microinjections; Minor Surgical Procedures; Modification; Molecular and Cellular Biology; Motor Neurons; Mouse Strains; Mus; Needles; Neural tube; Neurological Models; Neurons; Neurosciences; Nitrogen; Numbers; Operative Surgical Procedures; Optics; Phosphotransferases; Plasmids; Population; Preparation; Production; Prosencephalon; Reporter; Research; Research Infrastructure; Research Personnel; Resuscitation; Running; Services; Source; Stem cells; Tamoxifen; Time; Tissue Banking; Training; Transgenes; Transgenic Mice; Transgenic Organisms; Validation; Variant; Week; Work; animal facility; base; behavior test; blastocyst; design; embryonic stem cell; experience; foot; male; member; nestin protein; pathogen; recombinase; repository; research study; sperm cell; tissue culture; vector; vibration

c. Description The Biomedical Research Infrastructure (BRI) is an underground two floor, 120,000 square foot, 27,000 cage animal facility. This state-of-the-art automated facility is in the courtyard of the Biological Laboratories, which places it adjacent to the Northwest Building. The BRI houses a Genome Modification Facility (GMF) in an 800 square feet procedural room in the BRI with additional animal holding space for 1,000 cages in adjacent rooms. This room is equipped with 4 vibration free microinjection stations, micro-needle fabrication equipment, 4 banks of tissue culture incubators for embryo/ES cell culture, 3 tissue culture hoods, 3 laminar flow hoods for pathogen free minor surgeries, 12 stereomicroscopes of which 2 will be equipped with GFP optics and teaching video, automatic liquid nitrogen storage facilities, and computers. The GMF as a whole is governed by faculty committee of four individuals: Andrew McMahon (Molecular and Cellular Biology, Committee Chair), Joshua Sanes (CBS), and Kevin Eggan (HSCI). The committee oversees effective running of the GMF. The GMF staff consists of the following. ¿ The director, Manfred Baetscher, PhD, has broad experimental experience of genetic modification of mice and many years of managerial experience. He participates directly in production of genetically modified strains, remains current on emerging genetic approaches, and advises investigators on appropriate genetic strategies for their experiments. ¿ Transgenic research assistants: Two assistants, both with proven experience in either or both pronuclear injection of DMA and blastocyst injection and small animal surgery. ¿ Tissue culture technicians: A full time technician, soon to be hired will perform all aspects of ES cell culture, ES cell electroporation and clonal selection and DMA preparation forgenotyping. ¿ Animal husbandry: Two animal care technicians conduct all support functions for embryo generation and biopsy preparation. ¿ Administrative assistant: This individual is responsible for all ordering, billing and coordination between investigators and GMF. Finally, it is worth noting that the BRI will also contain behavioral testing suites equipped for phenotypic analysis of genetically modified mice, including models of neurological and psychiatric diseases. These will be supported from other sources, so are not described here, but it is our intention to integrate them with the GMF. The GMF provides the following services: ¿ Production of transgenic mice by pronuclear injection of DMA constructs in plasmids, BACs, or YACs ¿ ES cell targeting and injection to generate "knock-out" and "knock-in" mice ¿ Cryopreservation and storage of embryos and sperm for valuable strains not in current use ¿ Mouse strain resuscitation by in vitro fertilization and intracytoplasmic sperm injection (ICSI) ¿ Re-derivation of mouse strains by embryo transfer to render them pathogen-free ¿ The development of lentiviral vector based transgenes ¿ Consultation on genetic model creation ¿ Plans to offer gene targeting in embryonic stem cells and validation of DNA vectors and feeder lines We also hope the GMF will provide new functionality that we will add if this grant is funded, circumventing a bottleneck that many users of genetically modified mice encounter. There are a dozen or so strains that are highly useful for a broad variety of neuroscience applications. Most are publicly available but the waiting time to obtain them collaborators or stock centers (e.g., at Jackson Laboratories) is often 6 months or more, and the expense is considerable. Yet, few laboratories can afford to maintain stocks permanently of lines that may need only once every year or two. Lines in this category include: (a) Mice in which specific neuronal populations are marked with GFP or one of its spectral variants (e.g., motoneurons in YFP-16, layer 5 pyramids in YFP-H, very small numbers of forebrain neurons in GFP-M, or inhibitory interneurons in GAD65- GFP). (b) Mice in which expression of a reporter is conditional on expression of ere recombinase (e.g., Z/EG, Z/AP and thy1-stop-YFP). (c) Mice in which ere or flp recombinase is expressed in the neural tube generally (e.g., Nestin-Cre) or in specific populations of neurons (e.g., CaM kinase-Cre) or only following activation by tamoxifen (e.g., CAGS-CreER). So, we propose to maintain small colonies (approximately 4 cages of 4 males each) of each of about a dozen lines. The lines will be chosen by the Steering Committee. In most cases, the mice are already available in at least one user laboratory. Then, when any Harvard neuroscientist requires one of the lines, we will be able to provide two males within a week, and then replenish our own supply by breeding. We believe this repository will greatly decrease the barrier to use of valuable strains, thereby making possible risky or pilot experiments that would otherwise be prohibitively expensive or unduly slow.

c。描述 生物医学研究基础设施（BRI）是地下两层，120,000平方英尺，27,000笼 动物设施。这种最先进的自动化设施位于生物实验室的庭院中， 将其与西北大楼相邻。 BRI在800中设有基因组修饰设施（GMF） 布里的平方英尺程序室，附近有1000个笼子的额外动物持有空间 房间。该房间配备了4个无振动的微型注射站，微针制造设备， 4列胚胎/ES细胞培养的组织培养孵化器，3个组织培养引擎盖，3个层流罩 无病原体的次要手术，12个立体显微镜，其中2个将配备GFP光学元件和 教学视频，自动液氮存储设施和计算机。 GMF整体由四个人的教职委员会管理：Andrew McMahon（分子和 蜂窝生物学，委员会主席），约书亚·萨内斯（CBS）和凯文·埃格甘（HSCI）。委员会监督 GMF的有效运行。 GMF工作人员包括以下内容。 „董事Manfred Baetscher博士拥有广泛的实验经验 老鼠和多年的管理经验。他直接参与一般生产 修改后的菌株，在新兴的遗传方法上保持最新，并向研究人员提供建议 适合其实验的遗传策略。 �转基因研究助理：两名助理，均具有两者或两者的经验经验 DMA和胚泡注射和小动物手术的前注射。 �组织培养技术人员：一名全职技术人员，即将携带 培养，ES细胞电穿孔和克隆选择以及DMA制剂遗忘了。 „畜牧业：两名动物护理技术人员为胚胎生成所有支持功能 和活检准备。 »行政助理：此人负责所有订购，计费和协调 调查人员和GMF。 最后，值得注意的是，BRI还将包含相当于表型的行为测试套件 分析转基因小鼠，包括神经和精神病的模型。这些将是 从其他来源得到支持，因此在这里不描述，但我们的意图是将它们与GMF融为一体。 GMF提供以下服务： �通过质量注射DMA构建体中的DMA构建体，BAC或YACS的DMA构建体生产转基因小鼠 �ES细胞靶向和注射以产生“敲除”和“敲入”小鼠 »胚胎和精子的冷冻保存和存储，用于当前使用中的有价值菌株 »通过体外受精和胞质内精子注射（ICSI）通过体外受精和小鼠应变复苏 »通过胚胎转移对小鼠菌株的重新衍生以使其无病原体 �基于慢病毒载体的翻译的开发 »遗传模型创建咨询 »计划在胚胎干细胞中提供基因靶向靶向基因，并验证DNA矢量和较高的线条 我们还希望GMF将提供新的功能，如果这笔赠款是资助的，我们将添加这些功能，并规避 许多普遍修改的鼠标的用户遇到的瓶颈。有十几个菌株 对于广泛的神经科学应用非常有用。大多数是公开可用的，但等待时间 获得他们的合作者或股票中心（例如，在杰克逊实验室）通常是6个月或更长时间， 费用很大。然而，很少有实验室能够负担得起永久维护库存的线路 每年只需要一次。此类别中的线包括：（a）特定神经元的小鼠 种群用GFP或其光谱变体之一（例如，YFP-16中的运动神经元，第5层） YFP-H中的金字塔，GFP-M中的前脑神经元数量很少，或GAD65-中的抑制性神经元 GFP）。 （b）记者表达的小鼠是基于ERE重组酶表达的条件（例如z/eg， z/ap和thy1-stop-yfp）。 （c）在神经管中表达ERE或FLP重物组织酶的小鼠 （例如，Nestin-cre）或特定的神经元群体（例如CAM激酶-CRE）或仅在激活后才通过 他莫昔芬（例如Cags-Creer）。因此，我们建议维持小菌落（大约4个男性的4个笼子 每个）大约十二条线。这些线将由指导委员会选择。在大多数情况下， 至少一个用户实验室已经可以使用小鼠。然后，当任何哈佛神经科学家需要 其中之一，我们将能够在一周内提供两名男性，然后通过 选育。我们认为，该存储库将大大减少使用有价值的应变的障碍，从而使 否则可能会被禁止的冒险或试点实验昂贵或过低。