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 应用是分离和表征基因参与遗传性视网膜 退化影响动物和人类，重点是基因表达， 并确定相应基因产物的功能。的 如果我们想了解疾病发生的机制，后者至关重要 并设计了一些范例来尝试治愈它。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狗， 没有锥体。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） 目的是寻找那些在正常和非正常组织中差异表达的cDNA， 无视锥细胞视网膜一旦我们知道了这些cDNA在人体内的位置， 染色体，我们将确定相应的外显子/内含子边界 基因，并筛选他们的DNA突变的个人受影响， 视锥细胞营养不良或相关疾病。作为研究的例子， 在未来，我们分离出的视锥细胞基因，我们将描述， 确定视网膜裂素和Rp 1的功能， Xlrs 1和Rp 1基因。这些基因，最近在我们的 实验室检查，导致视网膜劈裂和adRP。我们将检验这个假设， 在从光感受器分泌后，视网膜劈裂素被摄取， 通过Muller细胞运输到内部视网膜。我们还将检查是否有一次 在内层视网膜中，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， 视网膜细胞结构的方式。关于Rp 1基因，我们成功地 在小鼠Rp 1基因座中引入人突变R677 X，获得嵌合的 动物，并确定有生殖系传播的突变Rpl 在他们的后代中。我们现在将描述形态学和 这一新的人类RP 1疾病动物模型的生理特征， 发展，并研究如何表达的Rpl突变基因的调控 体内的氧气。此外，我们将确定是否表达 突变基因只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 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，这是目前在我们的实验室。