Development of Genetic Tools to Study Retinal Regeneration

描述(申请人提供)：适应深色的白化斑马鱼在持续的强光下会失去杆状和锥状突起。光感受器是从内核层的成体干细胞群中再生出来的。在再生过程中，穆勒胶质细胞也可以增殖，或者去分化为神经元前体，或者转分化为光感受器。为了研究Muller胶质细胞在再生中的作用，有必要开发技术来调控转基因的时空表达，如Cre-lox位点特异性重组和Tet-on系统。虽然这两个系统都有效地用于小鼠遗传学，但都没有充分证明在转基因斑马鱼中发挥作用。我们将开发Cre-lox系统，以细胞特异性的方式表达报告(EGFP)，以检查细胞谱系。Cre酶将在GFAP或谷氨酰胺合成酶启动子的作用下在Muller胶质细胞中表达，它将催化第二个转基因中两个lox位点之间的重组。这将去除转录和翻译终止信号，并表达来自普遍存在的启动子的EGFP。因此，绿色荧光蛋白仅在穆勒胶质细胞及其衍生的任何细胞中表达。用免疫组织化学方法检测再生过程中不同时间点的视网膜，我们将明确识别再生过程中的EGFP阳性细胞和Muller胶质细胞的谱系。我们还将确定Tet-on系统在斑马鱼中是否起作用。我们将使用普遍存在的启动子或细胞特异性启动子来表达Tet反式激活因子(RTTA)。多西环素的加入将允许RTTA结合tre序列并激活EGFP报告基因的转录。证明Tet-on系统是一个紧密的可诱导系统，将使我们能够表达各种转基因，以检测内核层干细胞和Muller胶质细胞在再生反应中的作用。 相关性：调节转基因在斑马鱼中的时空表达将使我们能够在整个发育过程中检查基因的正常或突变形式的功能。我们将首先使用这些技术来研究放射状胶质细胞在光感受器再生中的作用。阐明Muller神经胶质细胞在斑马鱼光感受器再生中的作用可能会揭示诱导Muller神经胶质细胞再生人类视网膜退行性疾病的诱导性线索。

DESCRIPTION (provided by applicant): Dark-adapted albino zebrafish undergo loss of rods and cones when placed in constant intense light. The photoreceptors are regenerated from a population of adult stem cells in the inner nuclear layer. The Muller glia may also proliferate and either dedifferentiate into neuronal precursors or trans-differentiate into photoreceptors during regeneration. To examine the role of the Muller glia in regeneration, it is necessary to develop techniques to regulate the temporal-spatial expression of transgenes, such as the Cre-lox site- specific recombination and the Tet-On systems. While both of these systems are effectively used in mouse genetics, neither has been fully demonstrated to function in transgenic zebrafish. We will develop the Cre-lox system to express a reporter (EGFP) in a cell-specific manner to examine cell lineages. The Cre enzyme, which will be expressed in Muller glia using the GFAP or glutamine synthetase promoter, will catalyze the recombination between two lox sites in a second transgene. This will excise transcriptional and translational termination signals and express EGFP from a ubiquitous promoter. Thus, EGFP will only be expressed in Muller glia and any cells derived from these glia. Using immunohistochemical methods to examine retinas at different time points during regeneration, we will unambiguously identity the EGFP-positive cells and the lineage of the Muller glia during regeneration. We will also determine if the Tet-On system functions in zebrafish. We will express the Tet trans-activator (rtTA) using either an ubiquitous or cell-specific promoter. Addition of doxycycline will permit the rtTA to bind the TRE sequence and activate transcription of the EGFP reporter. Demonstrating that the Tet-On system is a tight inducible system will allow us to express a variety of transgenes to examine the roles of the inner nuclear layer stem cells and Muller glia in the regeneration response. Relevance: Regulating the temporal-spatial expression of transgenes in zebrafish will allow us to examine the function of either normal or mutant forms of a gene throughout development. We will initially use these techniques to examine the role of radial glia in the regeneration of photoreceptors. Elucidating the role of Muller glia in zebrafish photoreceptor regeneration may reveal inductive cues that could be used to induce Muller glia to regenerate photoreceptors in inherited human retinal degenerative diseases.