CORE--GENE KNOCKOUT

科学核心的总体目的是提供专业知识， 为实现三个项目的目标， 格兰特计划由于这些项目有许多共同的目标， 在核心单元中有效地执行常见的实验方法。 项目调查人员将有权使用核心设施， 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其中两个关键， 拟议项目。野生型和靶向ES细胞的分化 在一个项目和核心C中，体外也很重要。建立 这些程序的执行是劳动密集型的， 开发试剂和技术专长， 每个项目都可以单独进行。 因此，提出了一种敲除小鼠核心组件，以实现 计划/项目资助提案设定的目标。基因 提出的靶向/敲除小鼠核心组件将提供技术 在ES细胞衍生、靶向、处理和 操作和嵌合体生产的各个方面。然而，最初， 该核心将监督靶向载体的设计和构建 DNA最近发表的关于ets-1基因敲除小鼠的研究表明， 在桑椹胚-桑椹胚聚集体和ES细胞-野生型和ES细胞- 四倍体桑椹胚聚集可能是分析所必需的（69-72，79）。 核心董事专门研究胚胎致命突变体和逆行 根据我们最近的发现，我们的CTA-Fli 靶向突变体是一种隐性胚胎致死（项目2;新 初步数据）。ES细胞系的建立和核型分析， 维持原代饲养层成纤维细胞、条件培养基和ES 除了电穿孔，选择， 挑选和冷冻菌落。嵌合雄性产生包括 目标ES细胞系的扩增，胚胎供体的超数排卵， 桑椹胚分离和与ES细胞聚集，产生 输精管切除雄性和假性妊娠雌性，胚胎移植，以及 嵌合体的生长到断奶。嵌合雄性交配以鉴定 生殖系嵌合体和产生杂合子突变体的程度将是 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.