Photoreceptor Degeneration Assay-Transgenic Xenopus(RMI)

描述（由申请人提供）：这些研究的长期目标是寻找治疗光感受器变性的方法。虽然我们的大多数研究旨在了解光感受器变性的机制，以设计合理的治疗干预措施，但在本研究中，我们建议开发使用青蛙动物模型和高通量分子筛选（HTS）试验直接筛选潜在治疗方法的技术。我们已经开发了非洲爪蟾系，其中转基因既可以引起杆状光感受器以类似人类视网膜色素变性的方式退化，也可以报道光感受器在外部和活体动物中的生存能力。在本研究将要使用的这条线中，光感受器在生命的第二周发生退化，在此期间，通过透镜发出的荧光发生了超过一个数量级的变化。由于该系的成虫在一次繁殖中可以产生超过一千个后代，并且后代可以在光感受器退化期间饲养并在96孔板中进行检测，因此该系和类似系提供了很大的希望作为HTS检测的底物，旨在识别防止或减缓光感受器退化的化合物。在这个为期1年的项目中，我们提出优化蝌蚪在96孔板中的培养，优化冷血脊椎动物幼虫荧光成像屏幕成像平台，并进行中试筛选。除了具有发现治疗方法和研究光感受器生物学的工具的潜力之外，该屏幕将作为基于爪蟾或斑马鱼胚胎和幼虫的荧光报告构建未来神经退行性疾病HTS检测的原型。这些研究将利用约翰霍普金斯大学ChemCORE设施的化学文库和HTS基础设施。作为回报，我们希望能够提供工具和知识，以推进该设施未来的筛查。拟议的研究与促进人类健康有直接和间接的关系。直接相关的是，我们将专门筛选对患有视网膜变性的个体有益的治疗方法。虽然我们将使用的青蛙动物模型与视网膜色素变性最相似，这种疾病本身就折磨着全世界超过100万人，但我们希望，从我们的研究中得到的化学物质和信息将对更大一部分患有视力疾病的人产生影响。与人类健康的间接关联是，我们将有助于将高温高温分析带到活的脊椎动物有机体中，从长远来看，任何扩大高温高温分析范围的尝试都将有益于人类健康。

DESCRIPTION (provided by applicant): The long-term objective of these studies is to find therapies for the treatment of photoreceptor degenerations. Though most of our studies are aimed at understanding the mechanism of photoreceptor degeneration with the purpose of designing rational therapeutic interventions, in this study we propose to develop the technologies to directly screen for potential therapeutics using a frog animal model and a high throughput molecular screening (HTS) assay. We have developed lines of Xenopus laevis in which transgenes both cause rod photoreceptors to degenerate in a manner that resemble human retinitis pigmentosa as well as report the viability of photoreceptors externally and in live animals. In the line that will be used in this study, photoreceptors degenerate during the second week of life, during which period there is a change of fluorescence emitted through the lens of over an order of magnitude. Since adults of this line can produce over a thousand progeny in a single breeding, and the progeny can be raised and assayed in 96-well plates during the period of photoreceptor degeneration, this and similar lines offer great promise to serve as the substrates for HTS assays that aim to identify chemical compounds that prevent or slow photoreceptor degenerations. In this 1-year project, we propose to optimize the culturing of tadpoles in 96-well plates, optimize the imaging platform for fluorescence based imaging screens in cold-blooded vertebrate larvae, and perform a pilot screen. Besides having some potential to discover therapeutics and tools to study photoreceptor biology, this screen will serve as the prototype on which to build future HTS assays for neurodegenerative disorders based on fluorescent reporters using Xenopus or zebrafish embryos and larvae. These studies will take advantage of the chemical libraries and HTS infrastructure available at the Johns Hopkins ChemCORE facility. In return, we hope to be able to contribute tools and knowledge to advance future screens at the facility. The proposed studies have both direct and indirect relevance to promoting human health. The direct relevance is that we will be screening specifically for therapeutics that can be of benefit to individuals who suffer from retina degenerations. Though the frog animal model we will use most closely resembles retinitis pigmentosa, a disease which in of itself afflicts over 1 million people worldwide, we expect that both the chemicals and information that come out from our studies will have impact on a larger segment of those suffering from diseases of vision. The indirect relevance to human health is that we will be helping to bring HTS analyses to living vertebrate organisms, and any attempt to broaden the scope of HTS analyses will benefit human health in the long term.