We propose to develop a mouse model of human sickle cell anemia which faithfully reproduces the clinical features of this disease. These include anemia, hemolysis, sickling crises with infarction and end-organ damage involving the spleen, kidneys, lungs, liver, eyes, and central nervous system. We will use established procedures for generating transgenic mice to introduce human alpha and beta-S globin genes. Our approach will include the introduction of a yeast artificial chromosome (YAC) containing the whole beta-globin locus in which the beta-A gene is replaced by the beta-S gene, as well as human alpha globin genes that are expressed in a stage-specific manner. We will combine this approach with the inactivation of the endogenous murine beta globin locus and alpha globin genes by homologous recombination in embryonic stem (BS) cells. The inactivation of these genes will be accomplished by a combination of approaches, including targeted mutagenesis of the mouse beta-globin locus control region (LCR) and the alpha-globin upstream control element, as well as deletion of the structural genes. Adult mice derived from cross- breeding these lines will express hemoglobin S exclusively, reproducing the pattern of globin gene expression in SS patients. The generation of this murine model of sickle cell disease is essential for testing the therapeutic efficacy of the different strategies for gene therapy proposed in this program project grant. In addition, the model will be useful for studies of the pathophysiology of sickle cells, and for testing the effects of pharmacologic agents that may ameliorate clinical symptoms.
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