The hypothesis in this project is that Glut3 expression is critical for neuronal glucose supply through development, and that Glut3 plays an essential role in hypoxic-ischemic cellular apoptosis and necrosis. The primary goals are to determine the essential regulatory elements in the murine Glut3 gene that confer neuronal specificity, and susequently to determine the role of Glut3 in hypoxic-ischemic brain injury. First, the essential regulatory elements in the gene that confer neuronal specificity will be characterized by 5'-serial deletions of Glut3-1acZ reporter gene constructs, transiently transfecting the constructs into the mouse neuronal and nonneuronal cell lines, creating transgenic mice harboring Glut3-1acZ fusion genes to confirm the in vitro and in vivo functional role of the cis-elements that confer cell specificity, and to determine their role in hypoxic-ischemic brain injury. Second, the Glut3 gene will be overexpressed in neuronal cells and its role in apoptosis and necrosis determined. This will be done by stable transfection of Glut3 gene in neuronal cells and by generation of transgenic mice harboring multiple copies of Glut3 gene under the control of the neuron-specific synapsin I promoter. Third, neuron-specific Glut3 knockout mice will be generated using the Cre/LoxP recombination strategy to examine the influence of Glut3 ablation on hypoxic-ischemic brain injury.
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