[an error occurred while processing this directive]

Hereditas(Beijing) >

2017 , Vol. 39 >Issue 10: 930 - 938

DOI: https://doi.org/10.16288/j.yczz.17-080

Orginal Article

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

Expand
  • 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 date: 2017-03-06

  Revised date: 2017-04-07

  Online 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

Fold

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

Cite this article

Cuili Zhong, Guoling Li, Jianxin Mo, Rong Quan, Haoqiang Wang, Zicong Li, Zhenfang Wu, Xianwei Zhang . Effects of parameters, plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators[J]. Hereditas(Beijing), 2017 , 39(10) : 930 -938 . DOI: 10.16288/j.yczz.17-080

References

[1] 魏庆信, 郑新民, 乔宪风, 李莉, 华文君, 肖红卫, 王亚刚. 影响制备转基因猪效率的几个因素研究. 见: 亚洲实验动物学会联合会第三次会议暨中国实验动物学会第八届学术年会论文集. 北京, 2008.
[2] Liu TB, Dou HW, Xiang X, Li L, Li Y, Lin L, Pang XZ, Zhang YJ, Chen Y, Luan J, Xu Y, Yang ZZ, Yang WX, Liu H, Li FD, Wang H, Yang HM, Bolund L, Vajta G, Du YT. Factors determining the efficiency of porcine somatic cell nuclear transfer: data analysis with over 200, 000 reconstructed embryos. Cell Reprogr, 2015, 17(6): 463-471.
[3] Li XP, Yang Y, Bu L, Guo XG, Tang CC, Song J, Fan NN, Zhao BT, Ouyang Z, Liu ZM, Zhao Y, Yi XL, Quan LQ, Liu SC, Yang Z, G Ouyang HS, Chen YE, Wang Z, Lai LX. Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Res, 2014, 24(4): 501-504.
[4] Lai SS, Wei S, Zhao BT, Ouyang Z, Zhang QJ, Fan NN, Liu ZM, Zhao Y, Yan QM, Zhou XQ, Li L, Xin JG, Zeng YZ, Lai LX, Zou QJ. Generation of knock-in pigs carrying oct4-tdtomato reporter through CRISPR/Cas9-mediated genome engineering. PLoS One, 2016, 11(1): e0146562.
[5] Su WJ, Jiang H, Tang DS, Hong YH. Optimization of parameters of exogene transfection of Luchuan pig fetal fibroblasts in vitro mediated by liposome. Chin Agric Sci Bullet , 2011, 27(7): 300-303.
[5] 粟文俊, 蒋泓, 唐冬生, 洪亚辉. 脂质体转染巴马猪胎儿成纤维细胞条件的优化. 中国农学通报, 2011, 27(7): 300-303.
[6] 覃兆鲜, 潘天彪, 谢炳坤. 猪成纤维细胞转染方法的比较. 江苏农业科学, 2011, 39(3): 250-251.
[7] Liu W, Chen Y, Yue M, Yuan J, Qiu TW, Xiao D, Gu WW.An efficient method which can be used to transfect Tibetan minipig embryonic fibroblasts.Chin J Comparat Med, 2015(3): 64-66.
[7] 刘薇, 陈燕, 岳敏, 袁进, 邱添武, 肖东, 顾为望. 一种高效转染西藏小型猪胚胎成纤维细胞的方法. 中国比较医学杂志, 2015(3): 64-66.
[8] Lurquin PF. Gene transfer by electroporation. Mol Biotechnol, 1997, 7(1): 5-35.
[9] Ross JW, Whyte JJ, Zhao JG, Samuel M, Wells KD, Prather RS. Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts. Transgenic Res, 2010, 19(4): 611-620.
[10] Nakayama A, Sato M, Shinohara M, Matsubara S, Yokomine T, Akasaka E, Yoshida M, Takao S. Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection System?. Cloning Stem Cells, 2007, 9(4): 523-534.
[11] Richter A, Kurome M, Kessler B, Zakhartchenko V, Klymiuk N, Nagashima H, Wolf E, Wuensch A. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig. BMC Biotechnol, 2012, 12(1): 84.
[12] Tsuyama J, Bunt J, Richards LJ, Iwanari H, Mochizuki Y, Hamakubo T, Shimazaki T, Okano H. MicroRNA-153 regulates the acquisition of gliogenic competence by neural stem cells. Stem Cell Rep, 2015, 5(
Options
Outlines

/

〈 〉

www.chinagene.cn
备案号：京ICP备09063187号

版权所有 © 中国科学院遗传与发育生物学研究所 中国遗传学会
地址：北京市朝阳区北辰西路1号院　中国科学院遗传与发育生物学研究所2号楼B-208　
邮政编码：100101
联系电话：010-64807669
E-mail：yczz@genetics.ac.cn