CORE--MOUSE EMBRYONIC STEM (ES) CELL AND GENE TARGETING

该核心提供的服务形式多样，部分是为了满足 调查人员是用户。在许多情况下，服务是全面的，包括设计靶向构建体的建议，提供包含在构建体中的选择盒，电穿孔和ES集落的选择，DMA的制备，阳性克隆的核型分析，以及显微注射到胚泡中。在这些情况下，留给研究者的唯一任务是DNA印迹分析以鉴定靶向克隆。在其他情况下，特别是在靠近PI实验室的情况下，已向研究者提供了ES细胞，供其自己靶向。当通过Southern印迹分析鉴定出阳性克隆时，通过该Core进行核型分析和囊胚显微注射。除了基因靶向和胚泡注射的机制，该核心提供了一个额外的，宝贵的作用-建议和 协助分析小鼠表型。这主要是由藤原博士管理的。突变体的分析需要使用专门的方法，包括胚胎解剖、胚胎放血、来自卵黄囊、胎肝或外周血样品的造血集落测定、嵌合体分析和lacZ染色、原位杂交和组织学。该核心的经验和知识库为许多调查人员提供了非常有用的建议和实际帮助，其中一些人没有使用核心的其他服务。由于这些优势在波士顿地区已广为人知，因此整个哈佛医学区和其他地方的研究人员都向该核心寻求评估其小鼠敲除表型的指导。核心有三个同时的目标：首先，这个核心的存在允许为PI实验室的成员高效和成功地生产基因靶向小鼠。 第二，因为这个核心在人员和经验方面都有适当的专业知识，它为哈佛医学领域内外的许多研究人员提供服务。 第三，该核心将其产品（主要是小鼠品系）出口给其他地方的研究人员，以支持他们自己的研究项目，并在最广泛的意义上影响血液学领域。

Services provided by this Core take on various forms, in part tailored to meet the needs of the investigators who have been the users. In many instances, the service has been comprehensive and included advice on design of a targeting construct, providing selection cassettes for inclusion in the constructs, electroporation and selection of ES colonies, preparation of DMA, karyotype analysis of positive clones, and microinjection into blastocysts. Under these circumstances, the only task left to the investigator has been Southern blot analysis of DMA samples to identify targeted clones. In other instances, particularly with those in close proximity to the PI's laboratory, ES cells have been provided to the investigator for his/her own targeting. When positive clones are identified by Southern blot analysis, karyotyping and blastocyst microinjection are performed by this Core. Beyond the mechanics of gene targeting and blastocyst injection, this Core provides an additional, invaluable role--advice and assistance in the analysis of mouse phenotypes. this is largely managed by Dr. Fujiwara. The analysis of mutants requires use of specialized methods, including dissection of embryos, bleeding of embryos, hematopoietic colony assays from yolk sac, fetal liver, or peripheral blood samples, chimera analysis, and lacZ staining, in situ hybridization and histology. The experience and knowledge base of this Core has provided enormously useful advice and hands on assistance to many investigators, some who used no others services of the Core. Because the strengths became readily known in the Boston area, investigators throughout the Harvard Medical Area and elsewhere, approached this Core for guidance in evaluating their mouse knockout phenotypes. The Core has three simultaneous goals: First, the presence of this Core allows for highly efficient and successful production of gene targeted mice for members of the PI's laboratory. Second, because this Core had the appropriate expertise in both personnel and experience, it serves numerous investigators within the Harvard Medical Area and beyond. Third, this Core exports its products (largely mouse strains) to investigators elsewhere to support their own research projects and influence the field of hematology in the broadest sense.