遗传 ›› 2017, Vol. 39 ›› Issue (10): 930-938.doi: 10.16288/j.yczz.17-080

• 技术与方法 • 上一篇    下一篇

不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响

钟翠丽1(),李国玲1,莫健新1,全绒1,王豪强1,李紫聪1,吴珍芳1,2,张献伟1,2()   

  1. 1. 华南农业大学国家生猪种业工程技术研究中心,广州 510642
    2. 广东温氏食品集团股份有限公司,新兴 527400
  • 收稿日期:2017-03-06 修回日期:2017-04-07 出版日期:2017-10-20 发布日期:2017-09-14
  • 作者简介:钟翠丽,硕士研究生,专业方向：动物遗传育种与繁殖。E-mail: 1014942996@qq.com|张献伟，博士，在站博士后，研究方向：动物遗传育种与繁殖。E-mail: zxianw@163.com
  • 基金资助:
    国家转基因重大专项课题(2016ZX08006002);广东省科技厅平台条件建设项目(2013B070702003);粤西“扬帆计划”博士后人才扶持基金资助项目(2016)

Effects of parameters, plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators

Cuili Zhong1(),Guoling Li1,Jianxin Mo1,Rong Quan1,Haoqiang Wang1,Zicong Li1,Zhenfang Wu1,2,Xianwei Zhang1,2()   

  1. 1. National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guanzhou 510642, China
    2. Guangdong Food Group Limited Company, Xinxing 527400, China
  • Received:2017-03-06 Revised:2017-04-07 Online:2017-10-20 Published:2017-09-14
  • Supported by:
    the National Transgenic Major Project(2016ZX08006002);Department of Science and Technology of Guangdong Province(2013B070702003);Yuexi "Flying Sail Program" Postdoctoral Foundation

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摘要：

为获得猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs)最佳的电转染效率,本研究利用荧光激活细胞分选技术(fluorescence activated cell sorting, FACS)辅助优化NEPA 21和Nucleofector? 2b两种电转仪电转染PFFs细胞的参数,比较不同质粒用量和拓扑结构在ECM? 830、NEPA 21和Nucleofector? 2b中的转染效率。结果显示：NEPA 21电转PFFs的最佳穿孔参数为脉冲电压200 V,脉冲长度3 ms,脉冲间隔50 ms,脉冲次数3次,脉冲电压衰减幅度10%;Nucleofector? 2b在U-023的转染参数下达到最高转染效率。ECM? 830和Nucleofector? 2b的最适质粒用量都为10 μg,而NEPA 21为8 μg;超螺旋质粒比线性化质粒的转染效率更高,且3种仪器中Nucleofector? 2b转染效果最佳。本研究综合考虑电转仪、电转参数、质粒用量和拓扑结构的影响因素以优化PFFs的电转条件,为高效制备转基因猪及基因编辑猪的研究奠定基础。

关键词: 电转染, 猪胎儿成纤维细胞, ECM?830, NEPA 21, Nucleofector?2b

Abstract:

To obtain an ideal transfection efficiency of porcine fetal fibroblasts, fluorescence activated cell sorting (FACS) was used to optimize parameters for transfection of porcine fetal fibroblasts (PFFs) with ECM? 830, NEPA 21 and Nucleofector? 2b in different conditions such as electroporation parameters, plasmid dosages and topological structures. The results show that the optimum poring pulse parameter of NEPA 21 is voltage 200 V, continuous 3 ms, interval 50 ms, 3 times, voltage attenuation range of 10%; and the transfection efficiency of Nucleofector? 2b is highest under U-023 program. Under the optimum conditions, FACS analysis demonstrates that Nucleofector? 2b and ECM? 830 have the highest transfection efficiency when transfecting 10 μg supercoiled plasmids into PFFs, and 8 μg for NEPA 21. Supercoiled plasmids show higher transfection efficiencies than linearized plasmids. Moreover, Nucleofector? 2b has the highest transfection efficiency among the three electroporation instruments. This study paves the way to generate transgenic or gene editing pigs with high efficiency.

Key words: electroporation, porcine fetal fibroblasts(PFFs), ECM?830, NEPA 21, Nucleofector?2b