GENETIC BACKGROUND EFFECTS ON TRANSFERRED GAMMA GLOBIN GENES

了解胎儿珠蛋白合成的控制是直接相关的 因为镰刀中胎儿珠蛋白水平的增加而导致镰状细胞疾病 细胞病人与疾病的严重程度降低有关。 镰状细胞患者的胎儿珠蛋白水平升高。 有很强的遗传成分。与以下相关的遗传背景 在这项建议中研究的高胎儿珠蛋白水平是塞内加尔 β-珠蛋白基因簇的单倍型和与 X染色体。这项建议的目的是要深入了解 这些高胎球蛋白的形成与分子机制有关 表型。我们已经开发了一种利用培养的人红系 外周血造血祖细胞分析转珠蛋白 基因。该系统可用于研究胎儿珠蛋白的调节。 在本地环境中的序列，并潜在地为 镰状细胞病基因治疗的测试方法。监管 研究的序列将是G-伽马启动子、LCR和A-伽马增强子 来自塞内加尔单倍型的β-珠蛋白簇。使用的方法 将胎儿珠蛋白基因逆转录病毒转移到人外周血中 血液单个核细胞在无血清条件下的培养 才能产生红血球。基因转移的效率将被监测 对转基因红细胞进行定量聚合酶链式反应。的水平 转导基因在转染型红细胞中的表达 用定量逆转录聚合酶链式反应检测。的来源 这些研究的外周血将是男性和女性的镰状细胞 遗传背景与高或低相关的患者 胎儿球蛋白水平。拟议中的实验结果将 深入了解遗传因素与基因的作用之间的关系 控制胎儿珠蛋白合成的特定胎儿珠蛋白序列。

Understanding the control of fetal globin synthesis is directly relevant to sickle cell disease because increased levels of fetal globin in sickle cell patients are associated with a reduced severity of the disease. Elevated levels of fetal globin in sickle cell patients have been shown to have a strong genetic component. Genetic backgrounds associated with high fetal globin levels studied in this proposal are the Senegal haplotype of the beta-globin gene cluster and one linked to the X-chromosome. The aims of this proposal are to gain insights as to what molecular mechanisms are involved in establishing these high fetal globin phenotypes. We have developed a system using cultured human erythroid progenitors from peripheral blood for the analysis of transferred globin genes. This system allows for the study of fetal globin regulatory sequences in the native environment and potentially provide a basis for testing approaches to gene therapy of sickle cell disease. Regulatory sequences studied will be the G-gamma promoter, LCR, and A-gamma enhancer from a beta-globin cluster of the Senegal haplotype. Methodology used will be retroviral transfer of fetal globin genes into human peripheral blood mononuclear cells followed by culture of cells in serum-free medium to produce erythroblasts. Efficiency of gene transfer will be monitored by quantitative PCR of transfected erythroblasts. The level of transferred gene expression in transfected erythroblasts will be determined by quantitative reverse transcriptase PCR. The sources of peripheral blood for these studies will be male and female sickle cell patients with genetic backgrounds associated with either high or low levels of fetal globin. The results of the proposed experiments will provide insights into the relation of genetic factors to the role of specific fetal globin sequences in controlling fetal globin synthesis.