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一种优化的成年斑马鱼视网膜吗啡啉电转染方法

侯海涛¹, 吕金阳², 张志强², 陆颖³, 周翠萍³, 周天球³, 张书强¹, 徐绘^1,*

¹南通大学教育部和江苏省神经再生重点实验室，神经再生协同创新中心，南通 226001；²南通大学医学院，南通 226001；³南通大学教附属医院眼科，南通 226001

摘要

通过吗啡啉(morpholino, MO)电转染敲降斑马鱼视网膜内目标基因表达是研究基因功能的有效手段，但现有方法存在局限性。本文旨在发展一种优化的成年斑马鱼视网膜MO电转染方法。优化要点是在电极内侧涂抹超声凝胶，并使用方形电极而不是圆形电极。结果显示，使用超声凝胶可有效减少电转染所致视网膜损伤并简化实验步骤，采用方形电极显著提高了MO电转染效率。用本方法敲降视网膜再生相关基因Ascl1a显著抑制了视网膜祖细胞的生成。本方法是对现有MO电转染方法的优化。

关键词： 斑马鱼; 视网膜; 电转染; 吗啡啉

分类号：R339.14

An improved method for in vivo electroporation of morpholinos into the adult zebrafish retina

HOU Hai-Tao¹, LV Jin-Yang², ZHANG Zhi-Qiang², LU Ying³, ZHOU Cui-Ping³, ZHOU Tian-Qiu³, ZHANG Shu-Qiang¹, XU Hui^1,*

¹Co-innovation Center of Neuroregeneration, Ministry of Education and Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, China；²Medical School, Nantong University, Nantong 226001, China；³Department of Ophthalmology, Affiliated Hospital, Nantong University, Nantong 226001, China

Abstract

In vivo electroporation of morpholinos (MOs) into the retina of adult zebrafish is an efficient method to study gene function related to retinal disease and regeneration. However, the currently reported methods are complicated with low MO transfer efficiency and high probability to cause collateral damage. The present study was aimed to optimize the existing MO electroporation methods. Two major changes were made to MO electroporation procedure in zebrafish retina. One was to coat the inner side of the electrode with ultrasonic gel. The other was to replace the commonly used round electrode with novel rectangular one. The results showed that the use of ultrasonic gel reduced collateral damage caused by retinal electroporation and simplified the experimental procedure. The rectangular electrode significantly increased transfection efficiency of MO electroporation. In particular, knocking down the expression of Ascl1a in the retina by using our method significantly inhibited the generation of retinal progenitor cells. These results suggest our method is the optimization of the current MO electroporation methods and may be a better alternative for relevant researchers.

Key words: zebrafish;;;

收稿日期：2017-05-25　　录用日期：2018-01-11

通讯作者：徐绘　　E-mail: huixu82@126.com

DOI: 10.13294/j.aps.2018.0002

引用本文：

侯海涛, 吕金阳, 张志强, 陆颖, 周翠萍, 周天球, 张书强, 徐绘. 一种优化的成年斑马鱼视网膜吗啡啉电转染方法[J]. 生理学报 2018; 70 (1): 47-51.

HOU Hai-Tao, LV Jin-Yang, ZHANG Zhi-Qiang, LU Ying, ZHOU Cui-Ping, ZHOU Tian-Qiu, ZHANG Shu-Qiang, XU Hui. An improved method for in vivo electroporation of morpholinos into the adult zebrafish retina. Acta Physiol Sin 2018; 70 (1): 47-51 (in Chinese with English abstract).