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 镰状细胞病，因为镰状细胞中胎儿球蛋白水平的增加 细胞患者与疾病的严重程度降低有关。 镰状细胞病患者的胎儿珠蛋白水平升高 有很强的遗传成分 遗传背景与 高胎儿珠蛋白水平的研究，在这项建议是塞内加尔 单倍型的β-珠蛋白基因簇和一个连锁的 X染色体 本提案的目的是深入了解 分子机制参与了这些高胎儿珠蛋白的形成 表型。 我们已经开发了一个系统， 用于分析转移的珠蛋白的来自外周血的祖细胞 基因. 该系统允许研究胎儿珠蛋白调节 序列在天然环境中，并可能提供一个基础， 测试镰状细胞病基因治疗的方法。 监管 研究的序列将是G-γ启动子、LCR和A-γ增强子 来自塞内加尔单倍型的β-珠蛋白簇。 用方法 将胎儿珠蛋白基因逆转录病毒转移到人外周血 血液单核细胞，然后在无血清培养基中培养细胞 产生成红细胞。 将监测基因转移的效率 通过转染的成红细胞的定量PCR。 水平 在转染的成红细胞中的转移基因表达将是 通过定量逆转录酶PCR测定。 的来源 这些研究的外周血将是男性和女性镰状细胞 与高或低相关的遗传背景的患者 胎儿珠蛋白水平。 拟议实验的结果将 提供深入了解遗传因素的作用， 特异性胎儿珠蛋白序列控制胎儿珠蛋白合成。

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.