Probing photoreceptor cell biology using genetically modified amphibians.

使用转基因两栖动物探索感光细胞生物学。

• 批准号: RGPIN-2020-05193
• 负责人: Moritz, Orson
• 金额: $ 3.06万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740441
• 关键词: Probing; photoreceptor; cell; biology; using

The vertebrate retina contains two principle types of photoreceptor: rods and cones. Within these cells, photodetection occurs in an elaborate structure called the outer segment (OS), a modified sensory cilium. This structure contains many proteins unique to photoreceptors that are involved in light detection or OS structure. For many years, my laboratory has used genetically modified transgenic amphibians (specifically X. laevis) to study these cells due to the ease with which transgenic animals can be generated, as well as other advantages related to the large size of amphibian photoreceptors. Recently, we have developed CRISPR/Cas9 based techniques for gene knockout, and we recently generated knockouts of several photoreceptor-specific genes. We propose to use these animals, and to generate novel genetically modified animals, to study uncharacterized aspects of photoreceptor cell biology including: 1. Alternate pathways for generation of chromophore. We have found that knockout animals lacking RPE65, the isomerohydrolase thought to be responsible for generation of chromophore, still have a measurable electroretinogram. We will further characterize the visual responses of these animals, and in collaboration with Dr. A. Sampath at the Jules Stein Eye Institute, determine which cone subtypes are responsible. We will conduct further genetic manipulations to determine the nature of the pathway involved, and attempt to identify alterations in visual responses in animals in which this pathway is eliminated. 2: Physiological consequences of arrestin multimerization. We have knocked out the arrestin (SAG) gene resulting in animals with delayed dark adaptation. Some arrestins multimerize, although he physiological consequences of this are unclear. Rod arrestin is known to migrate between different cellular compartments in response to light. We will transgenically restore fluorescent arrestins to these animals that are primarily monomeric, dimeric, or multimeric. In collaboration with Dr. P. Calvert at SUNY we will use time-resolved fluorescence anisotropy microscopy to determine their multimeric states in vivo in different compartments, and determine the consequences for arrestin migration kinetics and dark adaptation kinetics in vivo. 3. Dissection of peripherin-2 functions. Peripherin-2 is a structural protein found at photoreceptor disk rims, where it creates and maintains curvature, while participating in a large number of proposed interactions. We have identified six peripherin-2 homologues in X. laevis. The presence of a large number of genes suggests that they may have developed specialized roles. By knocking out each peripherin-2 sequence separately, we may be able to dissect different unique aspects of peripherin-2 function. The large number of peripherin-2 sequences will likely make the resulting phenotypes less severe, and therefore potentially more structurally informative, than mammalian knockouts.

脊椎动物的视网膜包含两种主要类型的感光器：视杆细胞和视锥细胞。在这些细胞中，光检测发生在称为外节(OS)的精细结构中，这是一种改良的感觉纤毛。这种结构包含许多光感受器所特有的蛋白质，这些蛋白质参与光检测或OS结构。多年来，我的实验室一直使用转基因两栖动物(特别是莱维氏X.laevis)来研究这些细胞，因为转基因动物很容易产生，以及与两栖动物感光器大有关的其他优势。最近，我们开发了基于CRISPR/Cas9的基因敲除技术，并最近产生了几个光感受器特异基因的敲除。我们建议使用这些动物，并产生新的转基因动物，来研究光感受器细胞生物学的未知方面，包括：1.产生发色团的替代途径。我们已经发现，缺乏RPE65的基因敲除动物仍然有可测量的视网膜电信号。RPE65是一种被认为负责产生发色团的异麦芽水解酶。我们将进一步描述这些动物的视觉反应，并与Jules Stein眼科研究所的A.SamPath博士合作，确定哪些视锥细胞亚型是相关的。我们将进行进一步的基因操作，以确定所涉及的途径的性质，并试图确定该途径被消除的动物视觉反应的变化。2：芳香素多聚体的生理后果。我们已经敲除了arrestin(Sag)基因，导致动物的暗适应延迟。一些逮捕者使用多聚体，尽管其生理后果尚不清楚。已知Rod arrestin在不同的细胞隔间对光作出反应而迁移。我们将对这些主要是单体、二聚体或多聚体的动物恢复荧光阻滞剂。与纽约州立大学的P·卡尔弗特博士合作，我们将使用时间分辨荧光各向异性显微镜来确定它们在体内不同隔室中的多聚态，并确定在体内Arrestin迁移动力学和暗适应动力学的结果。3.对外周蛋白-2功能的剖析。外周蛋白-2是一种结构蛋白，发现于光感受器盘缘，在那里它创建和维持曲率，同时参与大量拟议的相互作用。我们已经在莱维氏X.laevis中鉴定了6个外周蛋白-2同源物。大量基因的存在表明，它们可能已经形成了专门的角色。通过分别敲除每个外周蛋白-2序列，我们可能能够剖析外周蛋白-2功能的不同独特方面。与哺乳动物基因敲除相比，大量的外周蛋白-2序列可能会使所产生的表型不那么严重，因此在结构上可能更具信息性。