Photoreceptor Degeneration Assay-Transgenic Xenopus(RMI)

描述(申请人提供)：这些研究的长期目标是寻找治疗光感受器退化的方法。虽然我们的大部分研究旨在了解光感受器退化的机制，目的是设计合理的治疗干预措施，但在本研究中，我们建议开发利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药物的技术。我们已经开发出非洲爪哇的品系，在这些品系中，转基因既能导致杆状感光细胞以一种类似于人类视网膜色素变性的方式退化，又能在外部和活体动物中报告感光细胞的生存能力。在这项研究中，光感受器在生命的第二周退化，在此期间，通过晶状体发射的荧光发生了超过一个数量级的变化。由于该品系的成虫可以在一次育种中产生1000多个后代，并且后代可以在感光细胞退化期间在96孔板中饲养和检测，因此该品系和类似品系很有希望作为HTS检测的底物，旨在识别防止或减缓感光细胞退化的化合物。在这个为期一年的项目中，我们建议优化96孔板中蝌蚪的培养，优化冷血脊椎动物幼虫基于荧光成像屏幕的成像平台，并进行中试筛选。除了有可能发现治疗方法和研究光感受器生物学的工具外，这一屏幕还将作为原型，利用非洲爪哇或斑马鱼胚胎和幼虫建立基于荧光记者的未来神经退行性疾病的HTS分析。这些研究将利用约翰霍普金斯化学中心现有的化学库和HTS基础设施。作为回报，我们希望能够贡献工具和知识来推动该设施未来的屏幕。拟议的研究对促进人类健康具有直接和间接的相关性。直接相关的是，我们将专门筛选对视网膜退化患者有益的治疗方法。尽管我们将使用的青蛙动物模型最接近于视网膜色素变性，这种疾病本身就困扰着全球100多万人，但我们预计，从我们的研究中得出的化学物质和信息将对更多患有视力疾病的人产生影响。与人类健康间接相关的是，我们将帮助将HTS分析带到活的脊椎动物有机体中，任何扩大HTS分析范围的尝试都将从长远来看有利于人类健康。

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.