大载体转染猪胎儿成纤维细胞的电转条件优化

doi:10.3864/j.issn.0578-1752.2019.03.013

摘要/Abstract

摘要：

背景随着生物技术发展,研究的生理机制和生物功能日益复杂,提高大载体的转染效率对多基因共表达系统、基因编辑技术、转基因育种等具有重要的意义。在转基因育种中,使用的转基因载体相对较大,而且转基因动物的制备效率也与供体细胞猪胎儿成纤维（porcine Fetal Fibroblasts,PFFs）细胞的转染效率有关。目的研究主要从转染参数、质粒用量和拓扑结构三方面,比较3种电转仪ECM ^?830/NEPA 21/Nucleofector ^TM2b的大载体转染效率,以探索大载体转染PFFs的最佳条件。方法使用3种不同电转仪将长达26 kb的携带增强型绿荧光蛋白基因的pPXAT-EGFP质粒转染1×10 ⁶个PFFs,48 h后使用流式细胞仪测定荧光细胞比例,从电转参数、质粒用量和拓扑结构三方面分别比较瞬时转染效率。结果首先比较电转仪不同参数的转染效率,结果显示当电转参数为脉冲电压300 V,脉冲长度1 ms,脉冲间隔50 ms,脉冲次数3次,NEPA 21转染PFFs的效率最高,为13.24%±1.63%,而Nucleofector ^TM 2b的最佳电转参数为U-023,其转染效率高达46.36%±3.95%。然后在最佳电转参数下分别比较6、8、10和12 μg的26 kb超螺旋质粒的转染效率,ECM ^?830和Nucleofector ^TM 2b转染PFFs的最佳质粒用量为12 μg,其转染效率分别为8.44%±0.90%（电转参数：脉冲电压300 V,脉冲长度1 ms,脉冲次数3 次）和14.63%±3.21%(电转参数：U-023),而NEPA 21使用10 μg质粒转染PFFs时效率达到最高（6.09%±0.72%）。最后比较不同质粒拓扑结构的转染效率,结果显示线性化质粒的转染效率较低,仅为超螺旋质粒转染效率的34.96%—48.39%。结论因此Nucleofector ^TM 2b转染PFFs的最佳条件为：U-023程序、12 μg超螺旋质粒;NEPA 21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、10 μg超螺旋质粒;ECM ^?830则在脉冲电压300 V,脉冲长度1 ms,脉冲次数3 次条件下转染12 μg超螺旋质粒可获得最佳转染效率。综合比较上述3种电转仪,26 kb大载体转染PFFs的最佳电转仪是Nucleofector ^TM 2b。

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

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

钟翠丽,李国玲,王豪强,莫健新,全绒,张献伟,李紫聪,吴珍芳,顾婷,蔡更元. 大载体转染猪胎儿成纤维细胞的电转条件优化[J]. 中国农业科学, 2019, 52(3): 530-538.

ZHONG CuiLi,LI GuoLing,WANG HaoQiang,MO JianXin,QUAN Rong,ZHANG XianWei,LI ZiCong,WU ZhenFang,GU Ting,CAI GengYuan. Optimizing the Electroporation Condition of Porcine Fetal Fibroblasts for Large Plasmid[J]. Scientia Agricultura Sinica, 2019, 52(3): 530-538.

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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	+

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