The major focus of this renewal application is to continue our ongoing studies on the regulation of rhodopsin gene expression. In addition, efforts are proposed to build on this work to develop an inducible photoreceptor specific promoter and also to define an efficient retinal pigment epithelium (RPE)-specific promoter. Transgenic mouse analysis of rhodopsin regulatory regions have defined both a rhodopsin proximal promoter region (RPPR) and a rhodopsin enhancer region (RER). The bovine minimal RPPR is sufficient to direct photoreceptor- specific gene expression, while the RER is important for high level expression. Through a combination of transient tranfection studies with primary chick retinal cell cultures, biochemical protein-DNA interaction assays, and in vitro transcription analysis, a number of DNA regulatory elements with the RPR have been defined, including binding sites for the neural retina leucine zipper (Nrl) and Ret 4 proteins. Using the yeast one-hybrid system, we have also cloned a number or novel homeodomain proteins which bind to the rhodopsin promoter. We propose to continue using these complementary approachers to more fully define and characterize the network of cis-acting elements and trans-acting factors which regulate rhodopsin transcription. the definition of rhodopsin promoter fragments that are active in transgenic mice has made possible studies in which exogenous or mutated proteins, such as dominant-negative forms of signaling receptors, are expressed in photoreceptor cells. Although this approach can be powerful, it is limited by the lack of control of the timing and expression level of the transgene. In order to address these limitations, we propose to develop an inducible and regulatable photoreceptor-specific promoter by combining the rhodopsin promoter with the reverse tetracycline transactivator system. We will first quantitatively assess the fidelity of the hybrid promoter system using a luciferase reporter assay. Then, we will generate transgenic mice carrying an inducible rds/peripherin cDNA construct in the background of the rds mutation so as to temporally and quantitatively assess how late in the degenerative process expression of the wild-type protein can lead to photoreceptor rescue. Due to the importance of the RPE in photoreceptor function and disease, the ability to modulate RPE gene expression in transgenic mice in a manner analogous to that possible with photoreceptor-specific promoters would clearly be desirable. In order to achieve this goal, the possible promoter activity of upstream regions from several genes that are expressed specifically in the RPE will be tested in cell culture and in transgenic mice.
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