CORE--GENE KNOCKOUT

科学核心的总体目的是提供专业知识 完成方案中三个项目的目标所必需的 格兰特项目。由于这些项目有许多共同的目标，因此 在核心单元中高效地执行常见的实验方法。 项目调查员将可以使用核心设施来运行他们自己的 实验，并将由核心主任和技术人员协助 关于实验设计、数据解释和技术 信息。基因敲除小鼠的产生是两个 建议的项目。野生型和靶向ES细胞的分化 体外培养在一个项目和核心C中也很重要。 而这些程序的执行是劳动密集型的，需要 开发试剂和技术专长，使其无法实现 每个拟议的项目单独进行都是可行的。 因此，提出了一种剔除鼠标核心组件来实现 计划/项目资助建议书设定的目标。基因 建议的靶向/击倒鼠标核心组件将提供技术 在ES细胞派生、定位、处理和管理的各个方面都有专业知识 操纵和嵌合体生产的方方面面。然而，最初， 该核心将指导靶向载体的设计和构建 DNA。最近发表的关于ETS-1基因敲除小鼠的文章证明了这种专业知识 在桑囊胚-桑囊胚聚集和ES细胞-野生型和ES细胞中- 分析可能需要四倍体桑椹胚聚集(69-72，79)。 核心董事在胚胎致命突变和逆行方面的专业知识 鉴于我们最近发现我们的CTA-Fli 靶向突变体是隐性胚胎致死(项目2；新 初步数据)。人胚胎干细胞系的建立及核型分析 原代饲养层成纤维细胞、条件培养液和胚胎干细胞的维持 除了电穿孔、选择、 采摘和冻结菌落。嵌合体雄性生产包括 靶向ES细胞系的扩增，胚胎捐献者的超数排卵， 桑囊胚的分离和与ES细胞的聚集、生成 输精管结扎男性和假孕女性，胚胎移植，以及 从嵌合体的生长到断奶。嵌合体雄性的交配以识别 生殖系嵌合体和产生杂合子突变体的程度将 核心参与。

The overall purpose of the Scientific Cores is to provide the expertise necessary to accomplish the goals of the three projects in the program Project Grant. Since these projects share many common goals, it is more efficient to perform common experimental approaches in the Core units. Project investigators will have access to core facilities to run their own experiments and will be assisted by the core director and technical staff concerning experimental design, data interpretation and technical information. The generation of knockout mice is central to two of the proposed projects. The differentiation of wild type and targeted ES cells in vitro is also important in one project and in Core C. The establishment and execution of these procedures is labor-intensive and requires the development of reagents and technical expertise that would not make it feasible for each of the proposed projects to be undertaken individually. As a result, a knockout mouse core component is proposed to achieve the goals set by the Program/Project Grant Proposal. The gene targeting/knockout mouse core components proposed will provide technical expertise in all aspects of ES cell derivation, targeting, handling, and manipulation and all aspects of chimera production. Initially, however, this core will supervise the design and construction of targeting vector DNAs. Recent publication on ets-1 knockout mice demonstrate that expertise in morula-morula aggregation and ES cell- wild type and ES cell- tetraploid morula aggregation may be necessary for analysis (69-72, 79). The core directors expertise with embryonic lethal mutants and retrograde analysis will be useful in light of our recent discovery that our CTA-Fli targeted mutant is a recessive embryonic lethal (Project 2; New Preliminary Data). The establishment and karyotyping of ES cell lines and the maintenance of primary feeder fibroblasts, conditioned medium, and ES cell lines are required in addition to electroporation, selection, picking, and freezing of colonies. Chimeric male production includes expansion of targeted ES cell lines, superovulation of embryo donors, morula isolation and aggregation with ES cells, the generation of vasectomized males and pseudo-pregnant females, embryo transfer, and growth of chimeras to weaning. Mating of chimeric males to identify germline chimeras and generate heterozygote mutants will be the extent of core involvement.