Human LDL receptor is expressed primarily in the liver and functions to clear the LDL and cholesterol from the circulation. Mutations of the LDL receptor have been shown to cause hypercholesterolemia which often results in cardiovascular diseases and myocardial infarction at an early age. The goal of this project is to use an inducible system to specifically target the human LDL receptor to the liver in transgenic mice as well as to study the efficacy of using adenoviral vector to deliver this inducible system into cell culture. The inducible system consists of a chimeric transcriptional regulator GLVP and a target gene containing the binding sites recognized by the regulator. In the presence of an inducer, RU486, the regulator binds and activates target gene expression. This inducible system has previously been demonstrated to effectively induce the expression of various target genes in cell culture and transgenic mice up to 5000-fold. To investigate controlled expression of human LDL receptor, we propose to: 1). Generate transgenic mouse target lines containing the human LDL receptor- cDNA; 2). Generate bitransgenic mouse lines capable of expressing the human LDL receptor in response to exogenous ligand; 3). Modify the regulator by substituting the viral activation domain with an activation domain from a human transcription factor to facilitate the future application of this inducible system for gene therapy; 4). Inducible expression of the LDL receptor in cultured cells using adenoviral delivery system. These studies are crucial in providing us with important kinetic data on LDL receptor expression and clearance of serum LDL and cholesterol. We expect that the results from this investigation shall enable us to formulate better protocols for future human gene therapy study in hypercholesterolemia and other cardiovascular diseases.
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