MOLECULAR MECHANISMS IN RETINAL DEGENERATIONS

描述(由申请人提供)：这项计划的长期目标 应用是分离和表征与遗传性视网膜有关的基因 影响动物和人类的退化，重点是在 并确定相应基因产物的功能。这个 如果我们想要了解一种疾病的机制，后者是必不可少的 是生产和设计范例，试图治愈它。为了获得表达的基因 在视锥细胞中，我们将利用成年后有视网膜的CD犬 没有球果的。我们将使用前几轮产品的微阵列 正常和成年CD犬视网膜代表性差异分析(RDA) 寻找在正常和正常组织中差异表达的cDNA 无视锥视网膜。一旦我们知道这些DNA在人类体内的位置 染色体上，我们将确定相应的外显子/内含子边界 基因，并筛查受感染个体的DNA突变 视锥细胞营养不良或相关疾病。作为我们将进行的研究的例子 在未来，随着我们分离的锥体基因，我们将表征和 测定视黄鞘蛋白和Rp1的功能，这是 Xlrs1和Rp1基因。这些基因，最近在我们的 实验室，导致视网膜劈裂和adrp。我们将检验这一假设 从光感受器分泌后，视黄醇被摄取并 由穆勒细胞运输到视网膜内侧。我们还将检查是否有一次 在视网膜内部，视黄醇参与了细胞的黏附、形成 在其他细胞表面与蛋白质的复合体，并在此 视网膜的细胞结构。关于Rp1基因，我们成功地 将人突变R677X引入小鼠Rp1基因座，获得嵌合 动物，并确定存在突变的RPL的生殖系传播 他们后代中的等位基因。我们现在将描述形态和 这一新的人类RP1病动物模型的生理特征 发展，并研究RPL突变基因的表达是如何调节的 通过体内的氧气。此外，我们还将确定是否 突变基因只影响带有突变等位基因的细胞或 RPL嵌合动物视网膜中邻近的野生型细胞。所有的 这些研究将使用最先进的分子生物学、遗传学、 细胞生物学和生物化学，这是目前在我们实验室的。

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.