MOLECULAR MECHANISMS IN RETINAL DEGENERATIONS

视网膜变性的分子机制

• 批准号: 6705056
• 负责人: DEBORA B FARBER
• 金额: $ 43.27万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6705056
• 关键词: animal genetic material tag; biotechnology; cone cell; dogs; electroretinography; fluorescent in situ hybridization; functional /structural genomics; gene expression; gene mutation; genetic disorder; genetic mapping; genetically modified animals; green fluorescent proteins; human tissue; laboratory mouse; messenger RNA; microarray technology; molecular cloning; molecular pathology; northern blottings; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; retina degeneration; rod cell; yeast two hybrid system

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.

描述（由申请人提供）：本项目的长期目标 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，重点是表达的基因 锥体，并确定相应基因产物的功能。这 如果我们想了解疾病发生的机制，后者至关重要 正在生产和设计范例来尝试解决它。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 狗 没有视锥细胞。我们将使用前几轮产品的微阵列 正常和成年 CD 狗视网膜的代表性差异分析 (RDA) cDNA 来查找在正常和正常情况下差异表达的 cDNA 无锥体视网膜。一旦我们知道了这些 cDNA 在人体中的位置 染色体，我们将确定相应的外显子/内含子边界 基因并筛选受影响个体的 DNA 突变 锥体营养不良或相关疾病。作为我们将进行的研究的例子 未来，我们将利用我们分离出的视锥细胞基因来表征和 确定视黄素和 Rp1 的功能，这两种蛋白质的产物 分别为 Xlrs 1 和 Rp1 基因。这些基因最近在我们的 实验室检查，引起视网膜劈裂和adRP。我们将检验以下假设： 从光感受器分泌后，视网膜劈裂素被吸收并 由米勒细胞转运至视网膜内层。我们还将检查是否有一次 在视网膜内部，视网膜劈裂素参与细胞粘附，形成 与其他细胞表面的蛋白质形成复合物并维持在该细胞中 视网膜细胞结构的方式。关于Rp1基因，我们成功地 在小鼠Rp1位点引入人类突变R677X，获得嵌合体 动物，并确定突变体 Rpl 存在种系传播 他们的后代中的等位基因。我们现在将表征形态和 这种新的人类RP1疾病动物模型的生理特征 发育，并研究 Rpl 突变基因的表达是如何调控的 通过体内的氧气。此外，我们将确定是否表达 突变基因仅影响具有突变等位基因的细胞，或者也影响具有突变等位基因的细胞 Rpl 嵌合动物视网膜中邻近的野生型细胞。所有的 这些研究将使用分子生物学、遗传学、 细胞生物学和生物化学，这是我们实验室的最新研究。