MOLECULAR AND GENETIC ANALYSIS OF A BLOOD CELL DISEASE

描述 （改编自申请人摘要）本项目的目标是使用 基因方法来确定最近发现的蛋白质的功能 在患有Wiskott-Aldrich综合征（WAS）的患者中发生突变， 以B细胞和T细胞免疫缺陷为特征的X连锁疾病， 湿疹血性腹泻和血小板减少症 将采用两种策略 来开发一个WAS的小鼠模型。 首先，他们瞄准并破坏了 小鼠胚胎干（ES）中人WAS基因的小鼠同源物 细胞产生WAS缺陷的干细胞。 为了快速分析 WAS缺乏对淋巴发育的影响，它们产生了 重组酶激活基因2缺陷型（RAG 2-/-）的体细胞嵌合体 互补系统 RAG 2缺陷小鼠无法重新排列 因此缺乏成熟的B和T 细胞 WAS缺陷型ES细胞已被引入RAG 2-/-胚泡中; 来自所得嵌合小鼠的所有B细胞和T细胞都是ES细胞 因此，可以选择性地分析WASp在淋巴细胞中的作用。 发展和功能。 初步研究表明，小鼠WAS- 淋巴细胞具有与人类疾病非常相似的缺陷。 WAS缺陷型ES细胞也将用于产生种系WAS-小鼠， 更广泛的影响和长期研究的分析。 将进行实验以确定生殖系是否 WAS缺陷型小鼠和WAS缺陷型RAG 2缺陷型嵌合体有效 研究人类疾病的模型。 将进行体内互补分析。 用WAS基因的不同区域进行，以定义和表征 蛋白质的功能结构域。 本申请是指导临床科学家发展奖， 申请人曾接受微生物学研究生培训， 免疫学，并已完成内科研究生培训 和临床胃肠病学。 申请人的长期目标是 建立并指导自己独立的基础研究项目 与影响人体免疫系统的疾病有关， 对影响粘膜免疫系统的疾病感兴趣。 因此，委员会认为， 这些研究是由弗雷德里克阿尔特博士共同赞助的， 遗传学在霍华德休斯医学研究所和血液中心 研究和博士丹尼尔波多尔斯基从胃肠单位在 马萨诸塞州总医院，都在哈佛医学院。 (End的 摘要）

DESCRIPTION (Adapted from applicant's abstract) The goal of this project is to use genetic approaches to determine the function of the protein recently found to be mutated in patients with the Wiskott-Aldrich Syndrome (WAS), a severe X-linked disease that is characterized by a B-and T-cell immunodeficiency, eczema, bloody diarrhea, and thrombocytopenia. Two strategies will be used to develop a mouse model of the WAS. First they have targeted and disrupted the murine homologue of the human WAS gene in murine embryonic stem (ES) cells to generate WAS-deficient stem cells. For a rapid analysis of the effects of the WAS-deficiency on lymphoid development, they have generated somatic chimeras in the recombinase activating gene-2-deficient (RAG2-/-) complementation system. RAG2-deficient mice are unable to rearrange immunoglobulin and T-cell receptor genes and therefore lack mature B and T cells. WAS-deficient ES cells have been introduced into RAG2-/- blastocyts; all of the B- and T-cells from resulting chimeric mice are of ES cell origin; thus one can analyze the role of WASp selectively in lymphocyte development and function. Preliminary studies suggest that murine WAS- lymphocytes have defects that closely resemble the human disease. WAS-deficient ES cells will also be used to generate germline WAS- mice for the analysis of more widespread effects and for long term studies. Experiments will be carried out to determine whether the germline WAS-deficient mice and the WAS-deficient RAG2-deficient chimeras are valid models to study the human disease. In vivo complementation analyses will be performed with various regions of the WAS gene to define and characterize functional domains of the protein. This application is for a Mentored Clinical Scientist Development Award to an Applicant who has received graduate training in microbiology and immunology, and has completed post graduate training in internal medicine and clinical gastroenterology. The applicant's long term goals are to establish and direct his own independent basic research program in studies related to diseases that affect the human immune system with clinical interests in diseases that affect the mucosal immune system. Accordingly, these studies are co-sponsored by Dr. Frederick Alt from the Department of Genetics at the Howard Hughes Medical Institute and the Center for Blood Research and by Dr. Daniel Podolsky from the Gastrointestinal Unit at the Massachusetts General Hospital, both at Harvard Medical School. (End of Abstract)