Development of Genetic Tools to Study Retinal Regeneration

描述（由申请人提供）：黑暗适应的白化病斑马鱼在置于恒定强光下时经历视杆细胞和视锥细胞的损失。光感受器是从内核层中的成体干细胞群体再生的。Muller胶质细胞也可以增殖，并且在再生期间去分化成神经元前体或转分化成光感受器。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节转基因的时空表达，例如Cre-lox位点特异性重组和Tet-On系统。虽然这两种系统都有效地用于小鼠遗传学，但都没有完全证明在转基因斑马鱼中发挥作用。我们将开发Cre-lox系统，以细胞特异性方式表达报告基因（EGFP），以检查细胞谱系。使用GFAP或谷氨酰胺合成酶启动子在Muller神经胶质中表达的Cre酶将催化第二转基因中两个lox位点之间的重组。这将切除转录和翻译终止信号并从普遍存在的启动子表达EGFP。因此，EGFP将仅在Muller神经胶质和源自这些神经胶质的任何细胞中表达。使用免疫组织化学方法检查视网膜在不同的时间点在再生过程中，我们将明确身份的EGFP阳性细胞和Muller胶质细胞的谱系再生过程中。我们还将确定Tet-On系统是否在斑马鱼中起作用。我们将使用普遍存在的或细胞特异性启动子表达泰特反式激活因子（rtTA）。多西环素的添加将允许rtTA结合TRE序列并激活EGFP报告基因的转录。证明Tet-On系统是一个紧密的诱导系统，将使我们能够表达各种转基因，以检查内核层干细胞和Muller胶质细胞在再生反应中的作用。 相关性：调节斑马鱼中转基因的时空表达将使我们能够检查基因在整个发育过程中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胶质细胞在斑马鱼感光细胞再生中的作用可能揭示可用于诱导Muller胶质细胞在遗传性人类视网膜变性疾病中再生感光细胞的诱导线索。

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.