Genome Modification Core

C.描述 生物医学研究基础设施(BRI)是一个地下两层，12万平方英尺，2.7万个笼子 动物设施。这个最先进的自动化设施位于生物实验室的院子里， 将其放置在西北建筑附近。BRI在800年中设立了基因组修改设施(GMF) 一带一路中有1,000个笼子的额外动物容纳空间，面积为1平方英尺 房间。这个房间配备了4个无振动微注射站，微针制造设备， 用于胚胎/胚胎细胞培养的4组组织培养孵化器，3个组织培养罩，3个层流罩 无病原体小手术，12台立体显微镜，其中2台将配备GFP光学元件和 教学录像、自动液氮储存设施和计算机。 转基因食品作为一个整体由四个人组成的教员委员会管理：安德鲁·麦克马洪(分子和 细胞生物学，委员会主席)，Joshua Sanes(哥伦比亚广播公司)，Kevin Eggan(HSCI)。该委员会负责监督 基因改造食品的有效运作。 基因改造食品的工作人员包括以下人员。 主任曼弗雷德·贝切尔博士在基因改造方面拥有丰富的实验经验。 会展和多年的管理经验。他直接参与了遗传基因的生产 转基因菌株，仍然是新出现的遗传方法的最新进展，并为研究人员提供建议 为他们的实验选择合适的遗传策略。 转基因研究助理：两名助理，两人都在其中一项或两项方面都有已证明的经验 原核注射DMA和囊胚注射及小动物手术。 ？组织培养技术员：即将招聘的全职技术员将对ES细胞进行全方位的操作 培养、ES细胞电穿孔、克隆选择和DNA分型准备。 ？畜牧业：两名动物护理技术员负责胚胎生成的所有支持职能 和活组织检查准备。 ？行政助理：该人员负责所有订单、帐单和协调 调查人员和转基因食品。 最后，值得注意的是，一带一路还将包含为表型配备的行为测试套件 转基因小鼠的分析，包括神经和精神疾病的模型。这些将是 其他来源的支持，所以不在这里描述，但我们的意图是将它们与转基因食品相结合。 基因改造食品提供以下服务： ？通过将DMA构建体原核注射到质粒、BAC或YAC中来产生转基因小鼠 ？ES细胞靶向和注射产生“敲除”和“敲入”小鼠 ？胚胎和精子的冷冻保存和储存，用于目前不使用的有价值的菌株 体外受精-卵胞浆内单精子注射(ICSI)复苏小鼠品系 通过胚胎移植重新衍生小鼠品系，使其无病原体 基于慢病毒载体的转基因研究进展 ？遗传模型创建咨询 ？计划在胚胎干细胞中提供基因靶向，并验证DNA载体和饲养线 我们还希望转基因食品将提供新的功能，如果这笔赠款得到资助，我们将添加这些功能，从而规避 许多转基因小鼠用户遇到的瓶颈。大约有十几个菌株是 对于广泛的神经科学应用非常有用。大多数都是公开的，但等待时间 获得他们的合作者或库存中心(例如，在杰克逊实验室)通常需要6个月或更长时间，并且 费用是相当可观的。然而，很少有实验室能够负担得起永久维持可能 每一两年只需要一次。这一类别的品系包括：(A)特定神经元的小鼠 用GFP或其光谱变体之一(例如，YFP-16第5层中的运动神经元)标记种群 YFP-H的锥体，GFP-M的极少量前脑神经元，或GAD65的抑制性中间神经元- GFP)。(B)报告的表达以ERE重组酶的表达为条件的小鼠(例如，Z/EG， Z/AP和TY1-STOP-YFP)。(C)ERE或flp重组酶在神经管中普遍表达的小鼠 (例如，巢蛋白-Cre)或在特定的神经元群体中(例如，CaM-KK-Cre)，或仅在通过 他莫昔芬(如CAGS-Creer)。因此，我们建议维持较小的群体(大约4个笼子，4个雄性 约12行中的每一行)。路线将由指导委员会选择。在大多数情况下， 至少有一个用户实验室已经有了老鼠。然后，当任何哈佛神经学家要求 其中一条线路，我们将能够在一周内提供两只雄性，然后补充我们自己的供应，通过 繁衍后代。我们相信这个资料库将大大降低使用有价值的菌株的障碍，从而使 可能的高风险或试验性实验，否则将耗资巨大或过于缓慢。

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.