Probing photoreceptor cell biology using genetically modified amphibians.

脊椎动物的视网膜包含两种主要类型的光感受器：视杆细胞和视锥细胞。在这些细胞中，光探测发生在一个称为外节（OS）的复杂结构中，这是一种经过修饰的感觉纤毛。这种结构包含许多光感受器特有的蛋白质，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X。laevis）来研究这些细胞，这是由于可以容易地产生转基因动物，以及与两栖动物光感受器的大尺寸相关的其他优点。最近，我们开发了基于CRISPR/Cas9的基因敲除技术，我们最近敲除了几个感光器特异性基因。我们建议使用这些动物，并产生新的转基因动物，研究感光细胞生物学的未表征的方面，包括：1。产生发色团的替代途径。我们发现，缺失RPE 65（被认为负责产生发色团的异构水解酶）的敲除动物仍具有可测量的视网膜电图。我们将进一步描述这些动物的视觉反应，并与A。朱尔斯·斯坦眼科研究所的Sampath博士，确定哪种锥细胞亚型负责。我们将进行进一步的遗传操作，以确定所涉及的途径的性质，并试图确定在动物的视觉反应中，该途径被消除的变化。2：抑制蛋白多聚化的生理后果。我们已经敲除了arrestin（SAG）基因，导致动物延迟黑暗适应。有些抑制蛋白会使细胞多聚化，但其生理后果尚不清楚。已知视杆细胞抑制蛋白响应于光而在不同细胞区室之间迁移。我们将转基因恢复荧光抑制蛋白，这些动物主要是单体，二聚体，或多聚体。与纽约州立大学的P. Calvert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体状态，并确定arrestin迁移动力学和体内暗适应动力学的后果。3. Peripherin-2功能的剖析。Peripherin-2是一种在感光盘边缘发现的结构蛋白，在那里它产生并保持曲率，同时参与大量的拟议相互作用。我们已经在X中鉴定了六个外周蛋白-2同源物。光滑。大量基因的存在表明它们可能已经发展出专门的作用。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。与哺乳动物敲除相比，大量的外周蛋白-2序列可能使所得表型不那么严重，因此可能在结构上提供更多信息。

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.