Thummel Ryan, Bailey Travis J, Hyde David R
Department of Anatomy, Wayne State University School of Medicine, USA.
J Vis Exp. 2011 Dec 27(58):e3603. doi: 10.3791/3603.
Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection. In all cases, resident Müller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days. Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.
许多毁灭性的遗传性眼病会导致进行性和不可逆转的失明，因为人类无法再生正在死亡或患病的视网膜神经元。相比之下，成年斑马鱼视网膜具有强大的自发再生能力，能够再生在各种不同视网膜损伤模型（包括视网膜穿刺、化学消融、集中高温和强光治疗）中丢失的任何神经元类型。我们实验室广泛研究了持续强光治疗后光感受器的再生以及玻璃体内注射哇巴因后视网膜内层神经元的再生。在所有情况下，视网膜中的穆勒胶质细胞重新进入细胞周期以产生神经祖细胞，这些神经祖细胞继续增殖并迁移到适当的视网膜层，在那里它们分化为缺失的神经元。我们描述了光损伤视网膜再生过程中的五个不同阶段，这些阶段以特定的细胞反应为特征。我们通过mRNA微阵列分析确定了视网膜再生每个阶段的几个差异表达基因。其中许多基因对眼睛发育也至关重要。为了测试每个候选基因/蛋白在视网膜再生过程中的作用，我们需要开发一种方法，有条件地仅在成年视网膜再生期间的特定时间限制候选蛋白的表达。吗啉代寡核苷酸被广泛用于研究斑马鱼、非洲爪蟾、鸡、小鼠发育过程中以及人类异种移植肿瘤中特定蛋白的功能丧失。这些修饰的寡核苷酸与互补RNA序列碱基配对，以阻断靶RNA的剪接或翻译。吗啉代在细胞中稳定，可以消除或“敲低”蛋白质表达三到五天。在这里，我们描述了一种在成年斑马鱼视网膜中有效敲低靶蛋白表达的方法。该方法采用标记有 Lissamine 的反义吗啉代寡核苷酸，将其注入成年斑马鱼眼的玻璃体中。然后使用电极镊子将吗啉代寡核苷酸电穿孔到背侧和中央视网膜的所有细胞类型中。Lissamine 为吗啉代寡核苷酸提供电穿孔所需的电荷，并且可以通过可视化来评估吗啉代寡核苷酸在视网膜细胞中的存在情况。视网膜中的条件性敲低可用于研究特定蛋白在再生过程中不同时间的作用。此外，这种方法可用于研究特定蛋白在未受损视网膜中的作用，例如在视觉转导和二级神经元的视觉处理等过程中。
