Photoreceptor Degeneration Assay-Transgenic Xenopus(RMI)

光感受器变性检测-转基因非洲爪蟾(RMI)

• 批准号: 7021338
• 负责人: NICHOLAS R MARSH-ARMSTRONG
• 金额: $ 18.3万
• 依托单位: HUGO W. MOSER RES INST KENNEDY KRIEGER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7021338
• 关键词: Xenopus; biotechnology; charge coupled device camera; chimeric proteins; drug discovery /isolation; drug screening /evaluation; embryo /fetus culture; eye disorder chemotherapy; gene expression; genetic models; genetically modified animals; green fluorescent proteins; high throughput technology; larva; retina degeneration; retinitis pigmentosa; small molecule; technology /technique development; visual photoreceptor

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.

描述（由申请人提供）：这些研究的长期目标是寻找治疗感光细胞变性的疗法。虽然我们的大多数研究旨在了解感光细胞变性的机制，目的是设计合理的治疗干预措施，在这项研究中，我们建议开发技术，直接筛选潜在的治疗方法，使用青蛙动物模型和高通量分子筛选（HTS）测定。我们已经开发了非洲爪蟾的线，其中转基因既导致杆光感受器退化的方式，类似于人类视网膜色素变性，以及报告的光感受器外部和活的动物的生存能力。在将用于本研究的线中，光感受器在生命的第二周期间退化，在此期间，通过透镜发射的荧光的变化超过一个数量级。由于该品系的成虫可以在单次繁殖中产生超过一千个后代，并且可以在光感受器变性期间在96孔板中饲养和测定后代，因此该品系和类似品系提供了很大的希望用作HTS测定的底物，HTS测定旨在鉴定防止或减缓光感受器变性的化合物。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的成像屏幕的成像平台，并进行中试筛选。除了有一定的潜力，发现治疗和工具来研究光感受器生物学，这个屏幕将作为原型，建立未来的HTS检测神经退行性疾病的基础上荧光报告使用非洲爪蟾或斑马鱼胚胎和幼虫。这些研究将利用约翰·霍普金斯化学核心设施提供的化学库和HTS基础设施。作为回报，我们希望能够贡献工具和知识，以推进该设施的未来屏幕。拟议的研究对促进人类健康有直接和间接的关系。直接相关的是，我们将专门筛选对视网膜变性患者有益的治疗方法。虽然我们将使用的青蛙动物模型与视网膜色素变性非常相似，这种疾病本身就困扰着全世界超过100万人，但我们希望我们研究中产生的化学物质和信息将对更大范围的视力疾病患者产生影响。与人类健康的间接关系是，我们将帮助将HTS分析带到活的脊椎动物生物体中，任何扩大HTS分析范围的尝试都将有利于人类健康。