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/NEPA21/Nucleofector^(TM)2b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12μg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12μg,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10μg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、12μg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、10μg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12μg超螺旋质粒可获得最佳转染效�
【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/NucleofectorTM 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/NEPA21/NucleofectorTM2b,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 NucleofectorTM 2b was U-023,whose transfection efficiency was 46.36%±3.95%.In addition,the transfection efficiencies of 6,8,10 and 12μg 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 12μg with 8.44%±0.90%efficiency(transfection parameters:pulse voltage 300 V,pulse length 1 ms,pulse number 3 times)and14.63%±3.21%(U-023),while the NEPA 21 achieved the highest efficiency of 6.09%±0.72%with 10μg plasmid.Finally,we compared the transfection efficiency of different quality topologies and fou
作者
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
ZHONG CuiLi;LI GuoLing;WANG HaoQiang;MO JianXin;QUAN Rong;ZHANG XianWei;LI ZiCong;WU ZhenFang;GU Ting;CAI GengYuan(College of Animal Science,South China Agricultural University/National Engineering Research Center for Breeding Swine Industry,Guangzhou 510642;Wenshi Food Group Co.Ltd.,Xinxing 527400,Guangdong)
出处
《中国农业科学》
CAS
CSCD
北大核心
2019年第3期530-538,共9页
Scientia Agricultura Sinica
基金
国家科技重大专项(2016ZX08006002)
国家自然科学基金(31802036)
广东省自然科学基金(2017A030310001)
