Genetic Analysis of Vertebrate Retinal Degeneration

描述：拟议的实验旨在利用脊椎动物遗传系统，能够识别影响斑马鱼视网膜的显性突变，特别强调与视紫红质运输有关的突变。该研究的长期目标包括鉴定与年龄相关性视网膜变性引起的失明相关的基因，以及表征视网膜变性的细胞和分子机制。引起显性遗传性夜盲症的突变将通过行为测定分离出来，并通过组织学和生理学方法加以证实。通过将表达编码红紫质-绿色荧光蛋白（GFP）融合蛋白的转基因斑马鱼与突变斑马鱼杂交，并通过共聚焦荧光显微镜检查后代，来评估正确的视紫质定位和运输。突变鱼的光感受器内的其他运输系统的附加特性将阐明细胞运输机制之间可能的相互作用。最后，鉴定负责这些突变的基因将通过定位克隆策略来实现。这项工作将为最终导致视网膜变性和与衰老相关的失明的光感受器细胞死亡的机制提供见解。

DESCRIPTION: The proposed experiments are designed to utilize a vertebrate genetic system capable of identifying dominant mutations affecting the zebrafish retina, with particular emphasis on mutations involved in rhodopsin trafficking. The long-term objectives of the proposed research include the identification of genes associated with blindness caused by age-related retinal degeneration and the characterization of the cellular and molecular mechanisms underlying retinal degeneration. Mutations causing dominantly inherited night blindness will be isolated by a behavioral assay and confirmed by histological and physiological methods. Proper rhodopsin localization and transport will be evaluated by breeding mutant zebrafish with transgenic zebrafish that express a transgene encoding a rhodopsin-green fluorescent protein (GFP) fusion protein and examining the progeny by confocal fluorescent microscopy. Additional characterization of other transport systems within the photoreceptors of mutant fish will elucidate possible interactions between cellular transport mechanisms. Finally, identification of the genes responsible for these mutations will be achieved by positional cloning strategies. The proposed work will provide insight to the mechanisms underlying photoreceptor cell death that ultimately lead to retinal degeneration and blindness associated with aging.