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，27,000笼 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， 将其放置在西北大厦附近。BRI在800个基因组中设有基因组修改设施（GMF） 在BRI中有一个平方英尺的手术室，在相邻区域中有额外的动物饲养空间，可容纳1，000个笼子 客房该房间配备有4个无振动显微注射站，微针制造设备， 4个用于胚胎/ES细胞培养的组织培养箱库，3个组织培养罩，3个层流罩， 无病原体的小手术，12台立体显微镜，其中2台将配备GFP光学器件， 教学录像、自动液氮储存设施和计算机。 GMF作为一个整体由四个人组成的教师委员会管理：Andrew McMahon（分子和 细胞生物学，委员会主席），约书亚萨内斯（哥伦比亚广播公司）和凯文埃根（HSCI）。该委员会负责监督 GMF的有效运行。 GMF的工作人员包括以下人员。 主任，曼弗雷德Baetscher，博士，具有广泛的实验经验的基因改造， 有多年的管理经验。他直接参与生产基因 改良的菌株，仍然是目前新兴的遗传方法，并建议研究人员 合适的遗传策略来进行实验。 转基因研究助理：两名助理，都在其中一个或两个方面都有经验 原核注射DMA和囊胚注射和小动物手术。 组织培养技术员：一名全职技术员，即将被雇用，将执行ES细胞的所有方面 培养、ES细胞电穿孔和克隆选择以及DNA制备用于基因分型。 畜牧业：两名动物护理技术人员负责胚胎生成的所有支持功能 和活检准备。 行政助理：此人负责所有订购，计费和协调之间 调查人员和转基因食品。 最后，值得注意的是，BRI还将包含为表型特征配备的行为测试套件。 分析转基因小鼠，包括神经和精神疾病模型。这些将是 由于其他来源的支持，因此在此不作描述，但我们打算将它们与转基因食品结合起来。 基因改造食品提供以下服务： 通过原核注射质粒、BAC或YAC中的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 冷冻保存和储存目前未使用的有价值的品系的胚胎和精子 通过体外受精和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的发展 基因模型创建咨询 计划在胚胎干细胞中提供基因靶向，并验证DNA载体和饲养细胞系 我们也希望GMF将提供新的功能，如果这笔赠款得到资助，我们将添加， 许多转基因小鼠用户遇到的瓶颈。有十几种菌株 对于广泛的神经科学应用非常有用。大多数是公开的，但等待时间， 为他们获得合作者或库存中心（例如，在杰克逊实验室）通常为6个月或更长时间， 费用相当可观。然而，很少有实验室能够负担得起永久性地维持可能 只需要一两年一次。这类细胞系包括：（a）小鼠，其中特定的神经元 用GFP或其光谱变体之一（例如，YFP-16第5层运动神经元 YFP-H中的锥体，GFP-M中的极少数前脑神经元，或GAD 65- GFP）。(b)其中报告基因的表达以ere重组酶的表达为条件的小鼠（例如，Z/EG， Z/AP和thyl-stop-YFP）。(c)在神经管中表达ere或flp重组酶的小鼠通常 (e.g., Nestin-Cre）或在特定的神经元群体（例如，CaM激酶-Cre）或仅在通过 他莫昔芬（例如，CAGS-CreER）。因此，我们建议维持小群体（约4笼，每笼4只雄性 每一个）的每一个约一打线。路线将由指导委员会选定。多数情况下 至少在一个用户实验室中已经有了小鼠。然后，当任何一个哈佛神经科学家要求 其中一条线，我们将能够在一周内提供两个男性，然后补充我们自己的供应， 育种我们相信，这个储存库将大大减少使用有价值菌株的障碍，从而使 可能的风险或试点实验，否则将过于昂贵或过于缓慢。

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.