Transgenic/Knock-out Mouse Shared Resource

转基因/基因敲除小鼠共享资源的主要目的是提供一个高效和 为梅西癌症中心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 基因组，或“敲除/敲入”小鼠，其中小鼠基因组中的内源基因是 特别是灭活或改良)已成为癌症研究中不可或缺的工具。的效用 这些模型的范围从研究肿瘤发生和发展的基本生物学到创造 用于评估新的治疗方法的基因准确的肿瘤模型。这个 转基因/基因敲除小鼠共享资源提供以下服务： 转基因小鼠生产：每个转基因核心至少产生三只创始小鼠。 敲出/敲入鼠标生产：内核可以执行以下全部或部分操作 方法：ES细胞电穿孔；在合适的选择条件下培养ES细胞；扩增 耐药克隆；克隆的聚合酶链式反应/Southern杂交筛选正确重组事件；重新电穿孔 使用ERE表达载体来删除漂浮的选择标记和随后的重新筛选 克隆；将定向ES细胞克隆注射到囊胚中用于嵌合小鼠的生产。 对于核心已执行ES细胞定位的项目，至少两个独立的ES细胞 克隆通常被注射到囊胚中，并至少产生五个嵌合体。 小鼠品系重建：通过胚胎移植获得感染病原体的小鼠品系，以产生 无特定病原体的小鼠。 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。 精子的氮气储存。当需要时，可以通过解冻来重建线路 精子和分离的WT雌性卵子的体外受精。 尾部DNA基因分型：核心分离来自尾部活检(或其他组织)的基因组DNA，由 研究人员并进行PCR基因分型。 咨询服务：核心为转基因/基因敲除小鼠提供多方面的咨询 技术，包括实验设计、媒介设计、鼠群维持预算，以及 准备向IACUC申请在研究中使用实验动物。此外， 核心员工提供关于群体整体管理的指导，包括繁殖策略、断奶、耳朵 拳打脚踢和尾巴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.