TRANSGENIC ANALYSIS OF PLATELET RECEPTOR EXPRESSION

描述 这一订正提案旨在说明 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， 糖蛋白受体 拟议的研究致力于改善 我们对GPIb-IX受体复合物的理解， 血管性血友病因子是血小板粘附所必需的， activation.在初步结果中，主要研究人员描述了 使用具有3 Kb的5'上游序列的6 Kb构建体， 两个外显子/一个内含子编码区的α亚基的GpIb和1 Kb的 3'下游序列，以产生表达α-半胱氨酸的转基因小鼠。 人血小板GpIb的β亚基相关的人血小板GpIb的β亚基 小鼠糖蛋白还提供了关于以下方面的初步数据： 人GpIb α亚基突变形式的表征， 在杂合子状态下导致Bernard Soulier综合征（巨大 微管网络有明显变化的血小板）。 根据上述初步结果，首席研究员 建议在五个具体领域完成工作。 第一调节 GpIb启动子的元件将通过定位起始位点来定义 启动子的5'端缺失， 通过转染到 佛波酯刺激的HEL细胞，并进行迁移率改变测定 用上述具有特定结构域的细胞的核提取物 如上所述确定。 第二，GpIb β和GpIX启动子 将根据关键监管区域进行表征， 然后与GpIb β、PF 4和GpIIa启动子进行比较， 确定共同监管领域。 第三，GpIb α启动子将 在转基因动物中进行评估，以确定是否可以使用 选择性表达异源蛋白如GpIIb/IIIa， 巨核细胞表面。 四、上述转基因动物将在 用于确定GpIb α是否在 非巨核细胞组织，如内皮细胞和细胞因子是否 诱导该基因产物合成增加。 第五，转基因 将产生具有上述GpIb α突变形式的动物 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 产生的异常由 完整巨核细胞/血小板的电子显微镜检查和 异常巨核细胞碎裂的体外研究。

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.