MOLECULAR MECHANISMS IN RETINAL DEGENERATIONS

描述（由申请人提供）：该项目的长期目标

DESCRIPTION (provided by applicant): The long-term objectives of this application are to isolate and characterize genes involved in inherited retina degenerations affecting animals and humans, with emphasis on genes expressed in cones, and to determine the function of the corresponding gene products. The latter is essential if we want to understand the mechanism by which a disease is produced and design paradigms to attempt its cure. To obtain genes expressed in cones, we will take advantage of the cd dog that by adulthood has a retina devoid of cones. We will use microarrays of the products of earlier rounds of representational differences analysis (RDA) of normal and adult cd dog retina cDNAs to find those cDNAs that are differentially expressed in normal and cone-less retinas. Once we know the location of those cDNAs in human chromosomes, we will determine the exon/intron boundaries of the corresponding genes and screen them for mutations in the DNA of individuals affected with cone dystrophies or related diseases. As examples of studies that we will carry out in the future with the cone genes that we isolate, we will characterize and determine the function of retinoschisin and Rp1, the protein products of the Xlrs 1 and Rp1 genes, respectively. These genes, recently isolated in our laboratory, cause retinoschisis and adRP. We will test the hypothesis that after being secreted from photoreceptors, retinoschisin is taken up and transported by Muller cells to the inner retina. We will also examine if once in the inner retina retinoschisin is involved in cell adhesion, forming complexes with proteins on the surface of other cells and maintaining in this way the retina cytoarchitecture. With regards to the Rp1 gene, we successfully introduced the human mutation R677X in the mouse Rp1 locus, obtained chimeric animals, and determined that there is germ-line transmission of the mutant Rpl allele in their progeny. We will now characterize the morphological and physiological features of this new animal model of human RP1 disease during development, and investigate how expression of the Rpl mutant gene is regulated by oxygen in vivo. In addition, we will determine whether expression of the mutated gene affects only the cells with the mutant allele or also the neighboring wild-type cells in the retinas of Rpl chimeric animals. All of these studies will use state-of-the-art methods in molecular biology, genetics, cell biology and biochemistry, which are current in our laboratory.