Retinal development, function, and disease are, to a significant degree, controlled by the pattern of genes expressed by the cells of the retina. In an effort to better understand the mechanisms regulating photoreceptor gene expression, we have been studying rhodopsin gene regulation as a model system. Using a variety of approaches, we, and others, have defined some of the DNA elements important in rhodopsin expression, have identified and cloned some of the transcription factors that bind to these DNA elements, and have shown that mutations in some of these factors can both interfere with normal photoreceptor development in the mouse and can cause retinal degeneration in man. This application for continued funding of these studies proposes to continue and broaden this work. The proposed work includes four broad aims: 1) efforts to more fully characterize already cloned factors and to continue using the yeast one-hybrid approach to clone additional factors that bind to the rhodopsin promoter; 2) initiation of studies analogous and complimentary to the rhodopsin work to study the promoters responsible for regulating expression of the cone opsins; 3) efforts to develop more efficient methods for transfection of primary photoreceptor cultures and to generate photoreceptor-like cell lines using inducible promoter technology to control expression of the SV40 TAg oncogene; and 4) use of custom retinal cDNA microarrays in conjunction of bioinformatic approaches as a complimentary approach to identify DNA regulatory elements and modules in the 5 '-upstream regions of genes that are preferentially expressed in photoreceptor cells. This last set of studies will be done in collaboration with Genomatix, a leader in the development of bioinformatics technology for promoter analysis.
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