Development of Genetic Tools to Study Retinal Regeneration

研究视网膜再生的遗传工具的开发

• 批准号: 7020845
• 负责人: David R Hyde
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7020845
• 关键词: Muller' s cell; albinism; biotechnology; gene expression; gene targeting; genetic promoter element; genetically modified animals; immunocytochemistry; nervous system regeneration; reporter genes; retinal adaptation; technology /technique development; zebrafish

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.

描述（由申请人提供）：当置于持续强光下时，适应黑暗的白化斑马鱼会失去视杆细胞和视锥细胞。光感受器由内核层中的成体干细胞群再生。穆勒胶质细胞也可能增殖并在再生过程中去分化为神经元前体或转分化为光感受器。为了研究 Muller 胶质细胞在再生中的作用，有必要开发调节转基因时空表达的技术，例如 Cre-lox 位点特异性重组和 Tet-On 系统。虽然这两个系统都在小鼠遗传学中有效使用，但都没有被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，以细胞特异性方式表达报告基因 (EGFP)，以检查细胞谱系。 Cre 酶将使用 GFAP 或谷氨酰胺合成酶启动子在 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将仅在米勒神经胶质细胞和源自这些神经胶质细胞的任何细胞中表达。使用免疫组织化学方法检查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 EGFP 阳性细胞和 Muller 胶质细胞的谱系。我们还将确定 Tet-On 系统是否在斑马鱼中发挥作用。我们将使用普遍存在的或细胞特异性的启动子来表达 Tet 反式激活子 (rtTA)。添加多西环素将允许 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多种转基因，以检查内核层干细胞和米勒胶质细胞在再生反应中的作用。 相关性：调节斑马鱼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正常或突变形式的功能。我们将首先使用这些技术来检查放射状胶质细胞在光感受器再生中的作用。阐明穆勒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线索，这些诱导线索可用于诱导穆勒神经胶质细胞在遗传性人类视网膜退行性疾病中再生光感受器。