解剖学报

解剖学报 ›› 2015, Vol. 46 ›› Issue (1): 133-137.doi: 10.16098/j.issn.0529-1356.2015.01.024

• 技术方法 • 上一篇    下一篇

一种自制电极鸡胚视顶盖转基因技术

杨慈清1, 2 李小英1 王聪睿2 李晗1 张必超1 林俊堂1,2*   

  1. 1.新乡医学院生命科学技术学院; 2. 河南省医用组织再生重点实验室，河南 新乡 453003
  • 收稿日期:2014-06-20 修回日期:2014-08-27 出版日期:2015-02-06 发布日期:2015-02-06
  • 通讯作者: 林俊堂 E-mail:yangciqing@126.com

A self-made electrode used for in vivo electroporation in chicken embryonic tectum

YANG Ci-qing 1,2LI Xiao-ying2 WANG Cong-rui1 LI Han1 ZHANG Bi-chao1 LIN Jun-tang 1,2*   

  1. 1. College of Life Science and Technology; 2. Key Laboratory of He’nan Province for Medical Tissue Regeneration, 2. Xinxiang Medical University, He’nan Xinxiang 453003，China
  • Received:2014-06-20 Revised:2014-08-27 Online:2015-02-06 Published:2015-02-06
  • Contact: LIN Jun-tang E-mail:yangciqing@126.com

摘要：

目的 建立一种高效鸡胚视顶盖电转基因技术，对自制电极进行评估。 方法 在鸡胚发育的第5天（E5），将0.5 g/L的pCAGGS-绿色荧光蛋白(GFP) 质粒0.25～0.5μl准确地注射到视顶盖内，每组60个鸡胚，在电压18V、每次脉冲60ms、间隔100ms、电脉冲6次的条件下，以原装电极为对照，自制电极为实验组进行定时定位活体电转基因，电转后在E6～E12时观察胚胎成活率，取材后在体视荧光显微镜下观察报告基因GFP表达结果以此判断转染的效率。结果 在鸡胚模型中，通过自制电极转染鸡胚视顶盖，24h后鸡胚成活率达到89%，在鸡胚发育到E12时存活率达到35%，与原装电极相比分别提高48.3%和600%个百分点。结论 自制电极在鸡胚视顶盖电转过程对胚胎的损伤小，转染后胚胎成活率高，为鸡胚视顶盖电转基因提供一种高效的转染技术。

关键词: 视顶盖, 自制电极, 活体电转, 鸡胚

Abstract:

Objective To establish an effectively transgenic technique for chicken embryo tectum electroporation by a self-made electrode. Methods Each embryo was precisely injected 0.25-0.5μl pCAGGS-green fluorescent protein (GFP)plasmid (0.5g/L) in the opitc tectum, and then preformed in vivo electroporation in a specific time and position using a self-made electrode as test group and an original electrode as control (60 eggs for earch group), under the conditions of 18 volt, each pulse 60ms, interval 100ms, and 6 times conditions. After electroporation, we analyzed the survival and positive ratio of embryo at E6-E12 using stereo fluorescence microscope to observe the reporter gene GFP expression and to judge the efficiency of transfection. Results The survival ratio of chicken embryo whose tectum was electroporated by the self-made electrode reach to 89% after 24 hours and 35% at E12, which were 48.3% and 600% times higher than original electrode. Conclusion This study demonstrates that the self-made electrode can cause tiny damage and ensure the higher survival ratio of electroporated chicken embryos so that it provides an effective transformation technique for in vivo electroporation in chicken embryonic tectum.

Key words: Opitc tectum, Self-made electrode, In vivoelectroporation, Chicken embryo