TRANSGENIC ANALYSIS OF PLATELET RECEPTOR EXPRESSION

这份修订后的提案旨在描述 巨核细胞生成、血小板生成和 通过分析血小板在体内的表达来研究其功能 糖蛋白受体。拟议的研究致力于改进 我们对血小板受体GPIB-IX受体复合体的认识 对于von Willebrand因子，这是血小板黏附和 激活。在初步结果中，首席调查员描述了 使用具有3kb的5‘上游序列的6kb的构建物， GPIBα亚基的2个外显子/1个内含子编码区 3‘下游序列用于产生表达α基因的转基因小鼠 人血小板膜糖蛋白Ib的亚基，与人血小板膜蛋白的β亚基相关 小鼠糖蛋白。还提供了关于以下方面的初步数据： 人GPIBα亚基突变型的鉴定 杂合子状态导致Bernard Soulier综合征(巨人 微管网络有明显变化的血小板)。 根据上述初步结果，首席调查员 建议完成五个具体领域的工作。第一个监管部门 GPIB启动子的元素将通过绘制起始位置来定义 启动子的5‘端缺失，以及 通过转染法评价各区域的转录效率 佛波酯刺激HEL细胞，并进行迁移率变化分析 用具有特定结构域的上述细胞的核提取液 如上所述确定的。第二，GPIB测试版和Gpix推动者 将针对关键监管区域和 然后与GPIB Beta、PF4和GpIIa启动子进行比较，以便 确定常见的监管领域。第三，GPIB阿尔法启动者将 在转基因动物身上进行评估，以确定它是否可以使用 选择性表达异源蛋白，如GPIIb/IIIa 巨核细胞表面。第四，上述转基因动物将被 用来确定GPIBα是否在 内皮等非巨核细胞组织与细胞因子 诱导该基因产物的合成增加。第五，转基因 将产生具有上述突变形式的GPIBα的动物 基因产物以确定该基因产物是否能够 通过异常相互作用诱发Bernard Soulier综合征 细胞骨架。对产生的异常情况进行检查。 完整巨核细胞/血小板的电子显微镜联合应用 异常巨核细胞体外碎裂的研究。

DESCRIPTION This revised proposal is directed at characterizing the molecular events of megakaryocytopoiesis, platelet production, and platelet function by analyzing the in vivo expression of platelet glycoprotein receptors. The proposed studies are devoted to improving our understanding of the GPIb-IX receptor complex, a platelet receptor for von Willebrand's factor which is essential for platelet adhesion and activation. In preliminary results, the Principle Investigator describes the use of a 6 Kb construct possessing 3 Kb of 5' upstream sequence, the two exon/ one intron coding region of alpha subunit of GpIb and 1 Kb of 3' downstream sequence to generate transgenic mice expressing the alpha subunit of human platelet GpIb associated with the beta subunit of the mouse glycoprotein. Preliminary data is also provided with regard to the characterization of mutant form of the human alpha subunit of GpIb which in the heterozygous state leads to Bernard Soulier syndrome (giant platelets with distinct changes in the microtubule network). Based upon the above preliminary results, the Principle Investigator proposes to complete work in five specific areas. First regulatory elements of the GpIb promoter will be defined by mapping the start site of the promoter, carrying out 5' deletions of the promoter and evaluating the transcriptional potency of regions by transfection into phorbal ester stimulated HEL cells, and conducting mobility shift assays with nuclear extracts of the above cells with specific domains identified as outlined above. Second, the GpIb beta and GpIX promoters will be characterized with regard to critical regulatory regions and then compared to the GpIb beta, PF4 and GpIIa promoters in order to identify common regulatory domains. Third, the GpIb alpha promoter will be evaluated in transgenic animals to ascertain whether it can be used to selectively express heterologous proteins such as GpIIb/IIIa on the surface of megakaryocytes. Fourth,the above transgenic animals will be employed to ascertain whether GpIb alpha is expressed in nonmegakaryocytic tissues such as endothelium and whether cytokines induce increased synthesis of this gene product. Fifth, transgenic animals will be generated with the above mutant form of the GpIb alpha gene product in order to ascertain whether this gene product is able to induce Bernard Soulier syndrome via abnormal interactions with cytoskeleton. The abnormalities produced with be examined by a combination of electron microscopy of intact megakaryocytes/platelets and in vitro studies of fragmentation of the abnormal megakaryocytes.