Transgenic/Knock-out Mouse Shared Resource

转基因/敲除小鼠共享资源的主要目的是提供高效且 为梅西癌症中心的成员生产转基因小鼠的经济手段。 转基因小鼠（包括转基因小鼠，其中定制的基因被引入到小鼠体内） 基因组，或“敲除/敲入”小鼠，其中小鼠基因组内的内源基因被 特别是失活或修饰的）已经成为癌症研究中不可或缺的工具。的效用 这些模型的范围从肿瘤发生和发展的基础生物学研究到 基因精确的肿瘤模型，用于评估新的治疗方法。的 转基因/敲除小鼠共享资源提供以下服务： 转基因小鼠生产：核心为每个转基因产生至少三只创始小鼠。 敲除/敲入小鼠生产：核心可以执行以下全部或部分功能： 方法：ES细胞电穿孔;在适当的选择条件下培养ES细胞;扩增ES细胞; 耐药克隆; PCR/Southern印迹筛选正确重组事件的克隆;再电穿孔 用ere表达载体删除floxed选择标记， 克隆;将靶向ES细胞克隆注射到胚泡中用于嵌合小鼠生产。 对于核心已经进行了ES细胞靶向的项目，至少两个独立的ES细胞 通常将克隆注射到胚泡中，产生最少5个嵌合体。 小鼠品系再衍生：通过胚胎移植对病原体感染的小鼠品系进行再衍生， 特异性无病原体小鼠。 精子冷冻保存：通过收集、冷冻和长期液体冷冻保存小鼠品系 精子的氮储存。当需要时，可以通过解冻来完成线路的重建 精子和进行从WT雌性分离的卵的体外受精。 尾DNA基因分型：核心从尾活检（或其他组织）中分离出基因组DNA， 并进行PCR基因分型。 咨询服务：核心提供转基因/基因敲除小鼠多方面的咨询 技术，包括实验设计，载体设计，小鼠群体维护预算， 准备向IACUC申请在研究中使用实验室动物。此外该 核心员工提供关于整个群体管理的指导，包括育种策略，断奶，耳 穿孔和尾部DNA分离。

The primary purpose of the Transgenic/Knock-out Mouse Shared Resource is to provide an efficient and economical means for producing genetically modified mice for members of the Massey Cancer Center. Genetically modified mice (including transgenic mice, in which customized genes are introduced into the genome, or "knock-out/knock-in" mice, in which endogenous genes within the mouse genome are specifically inactivated or modified) have become an indispensable tool in cancer research. The utility of such models ranges from studies of the basic biology of tumorigenesis and progression to the creation of genetically accurate tumor models for the evaluation of novel therapeutic approaches. The Transgenic/Knock-out Mouse Shared Resource offers the following services: Transgenic mouse production: The core generates at least three founder mice for each transgene. Knock-out/knock-in mouse production: The core can perform all or a subset of the following procedures: ES cell electroporation; ES cell culture under appropriate selection conditions; expansion of drug-resistant clones; PCR/Southern blot screening of clones for the correct recombination event; reelectroporation with a ere expression vector to delete floxed selection markers and subsequent rescreening of clones; injection of targeted ES cell clones into blastocysts for chimeric mouse production. For projects in which the core has performed the ES cell targeting, at least two independent ES cell clones are generally injected into blastocysts, and a minimum of five chimeras are generated. Mouse line rederivation: Lines of pathogen-infected mice are rederived by embryo transfer to generate specific pathogen-free mice. Sperm cryopreservation: Lines of mice are cryopreserved by collection, freezing, and long-term liquid nitrogen storage of sperm. When needed, re-establishment of the line can be accomplished by thawing sperm and performing in vitro fertilization of eggs isolated from WT females. Tail DNA genotyping: The core isolates genomic DNA from tail biopsies (or other tissue) provided by the investigator and performs PCR genotyping. Consulting services: The core provides consultation on many aspects of transgenic/knock-out mouse technology, including experimental design, vector design, budgeting for mouse colony maintenance, and preparation of applications to the IACUC for the use of laboratory animals in research. In addition, the core staff provides instruction on overall colony management, including breeding strategies, weaning, ear punching, and tail DNA isolation.