ADDUCIN ISOFORMS IN RBC AND PLATELET DIFFERENTIATION

红细胞和血小板分化中的内收肌蛋白异构体

• 批准号: 6411187
• 负责人: Diana Gilligan
• 金额: $ 21.91万
• 依托单位: BLOODWORKS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6411187
• 关键词: actins; cell differentiation; crosslink; erythrocytes; fluorescence microscopy; gene expression; gene targeting; genetic promoter element; genetically modified animals; human tissue; immunofluorescence technique; laboratory mouse; megakaryocytes; membrane proteins; messenger RNA; phosphorylation; platelet activation; platelet aggregation; platelets; polymerase chain reaction; protein isoforms; protein kinase C; protein structure function; spectrin; western blottings

The goal of this research is to test the hypothesis that the membrane skeleton protein adducin is critical to the assembly of the membrane skeleton during differentiation of erythrocytes and platelets. The membrane skeleton is crucial to the red cell, providing both support and flexibility as cells move rapidly through the circulation and traverse narrow capillaries. Defects in membrane skeleton proteins cause mild to severe hemolytic anemia and even hydrops fetalis. Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases. The membrane skeleton is also crucial to normal platelet function. The following specific aims are designed to analyze adducin's role in both erythrocyte and platelet differentiation and function: I. Determine the role of alternatively spliced adducins in erythrocyte differentiation a. complete the analysis of adducin expression patterns during normal human and mouse erythroid differentiation. b. elucidate the role of adducin in erythroid differentiation and function in vivo using two approaches: adducin null "knockout" mice and transgenic mice in which an erythroid specific promoter directs antisense mRNA production to block adducin expression. c. perform molecular dissection of the functions of the alternatively spliced isoforms in vivo by using an erythroid specific promoter to direct overexpression of tagged adducin isoforms (normal or mutant) to compete with normal adducin function in transgenic mice or to rescue knockout mice. II. Determine adducin's role in megakaryocyte differentiation and platelet function a. determine adducin expression patterns in mature platelets versus megakaryocytes b. determine the role of adducin in activation and aggregation of platelets c. elucidate the role of adducin in megakaryocyte differentiation and platelet function in vivo using two approaches: adducin null "knockout" mice and transgenic mice in which a platelet specific promoter directs antisense mRNA production to block adducin expression. d. perform molecular dissection of the functions of the alternatively spliced isoforms in vivo by using a platelet specific promoter to direct overexpression of tagged adducin isoforms (normal or mutant) to compete with normal adducin function in transgenic mice or to rescue knockout mice.

这项研究的目的是检验以下假设：膜骨架蛋白附加素对红细胞和血小板分化过程中膜骨架的组装至关重要。 膜骨架对红细胞至关重要，随着细胞在循环和狭窄的毛细血管中的循环迅速移动时，提供了支撑和柔韧性。 膜骨骼蛋白的缺陷会导致轻度至重度溶血性贫血甚至胎儿。 遗传性溶血性贫血（球状细胞增多症或椭圆细胞增多症）是最常见的遗传疾病之一。 膜骨架对于正常血小板功能也至关重要。 以下特定目的旨在分析Adducin在红细胞和血小板分化和功能中的作用：I。确定替代剪接的添加剂在红细胞分化中的作用a。在正常的人和小鼠红系分化过程中完成添加剂表达模式的分析。 b。使用两种方法：adducin null“基因敲除”小鼠和转基因小鼠在体内阐明了adducin在体内的作用，其中红细胞特异性启动子将反义mRNA产生引导以阻断adducin的表达。 c。通过使用红细胞特异性启动子直接过表达标记的Adducin同工型（正常或突变体），对体内剪接的同工型的功能进行分子解剖，以在转基因小鼠或挽救敲除小鼠中与正常的adducin功能竞争。 ii。确定添加剂在巨核细胞分化和血小板功能a中的作用a。确定成熟血小板与巨核细胞中的添加剂表达模式b。确定Adducin在血小板c的激活和聚集中的作用。使用两种方法阐明了adducin在体内的巨核细胞分化和血小板功能中的作用：adducin null“敲除”小鼠和转基因小鼠，其中小血小板特异性启动子将反义mRNA的产生引导以阻断Adducin的表达。 d。通过使用血小板特异性启动子直接过度表达标记的添加剂同工型（正常或突变体），对体内剪接的同工型的功能进行分子解剖，以在转基因小鼠中或拯救基因敲除小鼠在正常的附加蛋白功能中竞争。