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

这个核心所提供的服务有多种形式，部分是为了满足 曾是使用者的调查人员。在许多情况下，这项服务是全面的，包括以下方面的咨询：靶向构建物的设计、提供用于包含在构建体中的选择盒、电穿孔和选择ES克隆、制备DNA、阳性克隆的核型分析以及囊胚显微注射。在这种情况下，留给调查人员的唯一任务是对DMA样本进行Southern杂交分析，以确定目标克隆。在其他情况下，特别是在离PI实验室很近的情况下，已经向调查员提供了ES细胞，供他/她自己进行靶向。当通过Southern印迹分析鉴定阳性克隆时，该Core进行核型分析和囊胚显微注射。除了基因打靶和胚泡注射的机制外，这个核心还提供了一个额外的、宝贵的角色--建议和 协助分析小鼠的表型。这在很大程度上是由藤原博士管理的。突变的分析需要使用专门的方法，包括胚胎解剖、胚胎出血、卵黄囊、胎肝或外周血样本的造血细胞集落分析、嵌合体分析、LacZ染色、原位杂交和组织学。这个核心的经验和知识基础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。由于这种优势在波士顿地区很容易为人所知，整个哈佛医学区和其他地方的研究人员向该核心寻求指导，以评估他们的老鼠敲除表型。CORE有三个同步的目标：第一，这个CORE的存在使PI的实验室成员能够高效和成功地生产基因靶向小鼠。其次，因为这个核心在人员和经验方面都拥有适当的专业知识，它为哈佛医学区内外的众多研究人员提供服务。 第三，该中心将其产品(主要是老鼠品系)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.