Genetic Analysis of Vertebrate Retinal Degeneration

描述：拟议的实验旨在利用能够识别影响斑马鱼视网膜的显性突变的脊椎动物遗传系统，特别是涉及视紫红质运输的突变。这项拟议研究的长期目标包括识别与年龄相关性视网膜变性导致失明相关的基因，以及表征视网膜变性的细胞和分子机制。主要导致遗传性夜盲的突变将通过行为测试进行分离，并通过组织学和生理学方法进行确认。通过用表达视紫红质-绿色荧光蛋白(GFP)融合蛋白的转基因斑马鱼培育突变斑马鱼，并用共聚焦荧光显微镜检查后代，将评估适当的视紫红质定位和运输。突变鱼的光感受器内其他运输系统的额外特征将阐明细胞运输机制之间可能的相互作用。最后，将通过位置克隆策略来识别导致这些突变的基因。这项拟议的工作将为深入了解光感受器细胞死亡的潜在机制提供线索，这些死亡最终导致视网膜退化和与衰老相关的失明。

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.