MOLECULAR AND GENETIC ANALYSIS OF A BLOOD CELL DISEASE

描述 (改编自申请者的摘要)本项目的目标是使用 最近发现的确定蛋白质功能的遗传学方法 在Wiskott-Aldrich综合征(Was)患者中发生突变，这是一种严重的 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。将使用两种策略 建立风湿性关节炎的小鼠模型。首先，他们瞄准并破坏了 人的IS基因在小鼠胚胎干细胞中的小鼠同源物 细胞来产生基因缺失的干细胞。用于快速分析 对淋巴发育的影响，他们已经产生了 重组酶激活基因-2缺陷(RAG2-/-)的体细胞嵌合体 互补性制度。RAG2基因缺陷的小鼠无法重新排列 免疫球蛋白和T细胞受体基因，因此缺乏成熟的B和T 细胞。在RAG2-/-囊胚中引入了缺失的ES细胞； 所有来自嵌合小鼠的B细胞和T细胞都是ES细胞 因此，人们可以有选择地分析黄蜂在淋巴细胞中的作用 发展和功能。初步研究表明，小鼠- 淋巴细胞具有与人类疾病非常相似的缺陷。 Was缺陷的ES细胞也将被用于产生种系Was-小鼠 对更广泛影响的分析和长期研究。 将进行实验以确定生殖系是否 基因缺失的小鼠和基因缺失的RAG2嵌合体是有效的 研究人类疾病的模型。体内互补分析将是 与was基因的不同区域一起进行定义和表征 蛋白质的功能结构域。 此申请是为了获得临床科学家发展指导奖 曾接受微生物学研究生训练的申请人及 免疫学，并已完成内科研究生培训 和临床胃肠病学。申请者的长期目标是 建立并指导自己的独立基础研究项目 与影响人类免疫系统的疾病相关的临床 对影响粘膜免疫系统的疾病感兴趣。因此， 这些研究是由以下部门的Frederick Alt博士共同赞助的 霍华德·休斯医学院和血液中心的遗传学 研究和来自胃肠单位的Daniel Podolsky博士在 马萨诸塞州综合医院，两人都在哈佛医学院。(末尾 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)