Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (3): 530-538.doi: 10.3864/j.issn.0578-1752.2019.03.013

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Optimizing the Electroporation Condition of Porcine Fetal Fibroblasts for Large Plasmid

ZHONG CuiLi1,LI GuoLing1,WANG HaoQiang1,MO JianXin1,QUAN Rong1,ZHANG XianWei2,LI ZiCong1,WU ZhenFang1,2,GU Ting1(),CAI GengYuan1,2()   

  1. 1 College of Animal Science, South China Agricultural University/National Engineering Research Center for Breeding Swine Industry, Guangzhou 510642
    2 Wenshi Food Group Co. Ltd., Xinxing 527400, Guangdong
  • Received:2018-09-18 Accepted:2018-12-29 Online:2019-02-01 Published:2019-02-14

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Abstract:

【Background】 With the development of biotechnology, the physiological mechanisms and biological functions of research are becoming more and more complex, and improving the transfection efficiency of large vectors is of great significance for multi-gene co-expression systems, genome editing, and transgenic breeding. In transgenic breeding, the transgenic vectors is relatively large, and the efficiency of generating transgenic animals is also attributed to the transfection efficiency of porcine fetal fibroblasts (PFFs). 【Objective】 Therefore, this study mainly compared the electroporation efficiency of three electroporation apparatus ECM ? 830/NEPA 21/Nucleofector TM 2b on various parameters, plasmid dosage and topology, to explore the optimal electroporation condition of large vector in PFFs. 【Method】 We transfected a 26 kb plasmid, namely pPXAT-EGFP, into the PFFs, and then the electroporation efficiencies of various parameters of three electroporation apparatus ECM? 830/NEPA 21/Nucleofector ? 2b, and the dosage and topological structure of the plasmid by the flow cytometry were compared. 【Result】 By comparing the transfection efficiency of different transfection parameters, the results showed that the highest transfection efficiency of NEPA 21 was 13.24%±1.63% at pulse voltage 300 V, pulse length 1 ms, pulse interval 50 ms, pulse number 3 times, and the optimal electrical rotation parameter of Nucleofector TM 2b was U-023, whose transfection efficiency was 46.36%±3.95%. In addition, the transfection efficiencies of 6, 8, 10 and 12 μg 26 kb supercoiled plasmids were compared under the optimal electroporation parameters, and the results showed that the optimal plasmid dosage for ECM? 830 and Nucleofector TM 2b transfected PFFs was 12 μg with 8.44% ± 0.90% efficiency (transfection parameters: pulse voltage 300 V, pulse length 1 ms, pulse number 3 times) and 14.63% ± 3.21% (U-023), while the NEPA 21 achieved the highest efficiency of 6.09% ± 0.72% with 10 μg plasmid. Finally, we compared the transfection efficiency of different quality topologies and found that the transfection efficiency of linearized plasmids was low, only 34.96%-48.39% of the supercoiled plasmids. 【Conclusion】 Therefore, the optimal electroporation condition of PFFs by Nucleofector ? 2b was U-023 procedure, 12 μg supercoiled plasmid; NEPA 21 was pulse voltage 200 V, pulse length 3 ms, pulse interval 50 ms, pulse number 3 times, and 10 μg supercoiled plasmid; ECM? 830 was transfected with 12 μg supercoiled plasmid at pulse voltage 300 V, pulse length 1 ms, and pulse number 3 times to obtain the highest transfection efficiency. Comprehensive comparison of the above three kinds of electroporation apparatus, the optimum for transfecting PFFs with 26 kb large carrier was Nucleofector ? 2b.

Key words: electroporation, large plasmid, porcine fetal fibroblasts, ECM ?830, NEPA 21, Nucleofector ? 2b

Fig. 1

The plasmid profile of pPXAT-EGFP"

Table 1

Poring pulse of NEPA21"

编号
Number		 脉冲电压
Voltage (V)		 脉冲长度
Length (ms)		 脉冲间隔
Continuous (ms)		 脉冲次数
Times		 衰减幅度
Voltage attenuation range (%)		 电极
Electrode
NEPA-1 150 5 50 2 10 +
NEPA-2 200 3 50 3 10 +
NEPA-3 275 1.5 50 2 10 +
NEPA-4 300 1 50 3 10 +

Table 2

Grouping of optimizing the parameters of Nucleofector? 2b"

组别
Group		 电转参数
Parameters		 电转预混液
Transfection mixture
处理组1 Group 1 LONZA-A1) 100 μL Nucleofector? 2b电转液3)+8 μg质粒
100 μL the transfection mixture of Nucleofector? 2b and 8 μg plasmid
处理组2 Group 2 LONZA-U2)
空白组Blank group 不电转Non transfection 100 μL Nucleofector? 2b电转液Only 100 μL the transfection mixture of Nucleofector? 2b

Fig. 2

The transfection efficiency of different parameters A: Comparison of transfection efficiency between 4 kinds of NEPA21 electroporation parameters which are shown in Table 2; B: Comparison of transfection efficiency between two kinds of Nucleofector? 2b electroporation parameters, LONZA-A: A-033; LONZA-U: U-023o"

Fig. 3

The transfection efficiency of plasmid with different dosages BTX: The better parameter of ECM? 830（voltage 200 V, continuous 3 ms and 3 times）;NEPA-4: The better parameter of NEPA 21(voltage 200 V, continuous 3 ms, interval 50 ms and 3 times); LONZA-U: The better parameter of Nucleofector? 2b(U-023)"

Fig. 4

The transfection efficiency of plasmid with different topological structures A: Cells transfected with the different topological structures of plasmid were observed by fluorescence microscopy; B: Comparison of transfection efficiency between supercoiled plasmid and linearized plasmid. BTX: The better parameter of ECM? 830(voltage 200 V, continuous 3 ms and 3 times); NEPA-4: The better parameter of NEPA 21(voltage 200 V, continuous 3 ms, interval 50 ms and 3 times); LONZA-U: The better parameter of Nucleofector? 2b(U-023); Sp: Supercoiled plasmid; Lp: Linearized plasmid.*: P<0.05; **: P<0.01"

Fig. 5

The transfection efficiency of different electroporation apparatus BTX: The better parameter of ECM? 830 (voltage 200 V, continuous 3 ms and 3 times); NEPA-4: The better parameter of NEPA 21(voltage 200 V, continuous 3 ms, interval 50 ms and 3 times); LONZA-U: The better parameter of Nucleofector? 2b(U-023)"

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