Probing photoreceptor cell biology using genetically modified amphibians.

脊椎动物的视网膜包含两种主要的感光细胞：视杆细胞和视锥细胞。在这些细胞内，光探测发生在一种叫做外段（OS）的复杂结构中，这是一种改良的感觉纤毛。这种结构包含许多光感受器特有的蛋白质，这些蛋白质参与光探测或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，因为转基因动物很容易产生，而且两栖动物的光感受器体积大。最近，我们开发了基于CRISPR/Cas9的基因敲除技术，并且我们最近产生了几个光受体特异性基因的敲除。我们建议利用这些动物，并产生新的转基因动物，来研究感光细胞生物学的未表征方面，包括：1。生色团产生的交替途径。我们发现，缺失RPE65的基因敲除动物仍然具有可测量的视网膜电图。RPE65是一种被认为负责发色团生成的异构体水解酶。我们将进一步表征这些动物的视觉反应，并与朱尔斯·斯坦眼科研究所的a·萨姆帕斯博士合作，确定哪些锥体亚型负责。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，并试图确定该途径被消除后动物视觉反应的变化。2：多聚性阻滞的生理后果。我们已经敲除了阻滞蛋白（SAG）基因，导致动物对黑暗的适应延迟。有些逮捕是多重的，尽管其生理后果尚不清楚。杆状细胞阻滞蛋白响应于光在不同的细胞间迁移。我们将通过转基因恢复这些主要是单体、二聚体或多聚体的动物的荧光抑制。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间中的多聚态，并确定体内阻滞迁移动力学和暗适应动力学的结果。3. 外周蛋白-2功能解剖。外周蛋白-2是一种在光感受器圆盘边缘发现的结构蛋白，它在那里产生并保持曲率，同时参与大量的相互作用。我们已经鉴定出6个外周蛋白-2同源物。大量基因的存在表明它们可能具有特殊的作用。通过分别敲除每个外周蛋白-2序列，我们可能能够解剖外周蛋白-2功能的不同独特方面。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，因此可能比哺乳动物基因敲除更具有结构信息性。

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.