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.