绵羊成纤维细胞OAR-L1高效转染方法的探索  

Exploration of high-efficiency transfection methods for sheep fibroblasts OAR-L1

作　　者：吴飞 吴杰 陈学秋[1] 周静茹 张惠 黄艳 时恒枝 杨怡 马光旭 杜爱芳[1] WU Fei;WU Jie;CHEN Xueqiu;ZHOU Jingru;ZHANG Hui;HUANG Yan;SHI Hengzhi;YANG Yi;MA Guangxu;DU Aifang(Institute of Preventative Veterinary Sciences,College of Animal Sciences,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学动物科学学院动物预防医学研究所,杭州310058

出　　处：《浙江大学学报（农业与生命科学版）》2022年第1期96-105,共10页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家重点研发计划(2017YFD0501203);国家自然科学基金(32002304);浙江省公益技术研究计划(LGN18C180002)。

摘　　要：为了在绵羊肺成纤维细胞OAR-L1中高效表达外源蛋白,探索适合该细胞的转染方法,本研究比较了聚乙烯亚胺(polyethyleneimine,PEI)、脂质体2000转染试剂(Lipofectamine^(TM) 2000 transfection reagent,Lipo 2000)、成纤维细胞转染试剂盒(Cytofect^(TM) fibroblast transfection kit,CF2)以及慢病毒介导的细胞转染方法在OAR-L1细胞中表达外源蛋白的效果。结果显示:以pLentiCMV-EGFP-Puro或pLentiCMV-mCherry-Puro荧光质粒转染OAR-L1细胞时,细胞密度和转染试剂用量均可影响PEI、Lipo 2000和CF2介导的细胞转染效率,且三者最佳转染效率均不超过30%,而慢病毒介导的细胞侵染不受细胞密度和病毒液用量的限制,获得的荧光细胞数目也显著高于前三者。包装好的病毒液可以在4或-80℃条件下至少保存15 d而侵染效果不受影响。在OAR-L1细胞中共表达2种外源蛋白时,包装获得的2种慢病毒液等比例混合后再侵染的方式能获得更高的共转率。综上所述,慢病毒侵染是一种能够实现在OAR-L1细胞中高效表达外源蛋白的细胞转染方式,本研究结果为其他难转染细胞系转染方式的选择提供了一定的参考。In order to achieve high-efficiency expression of exogenous protein in sheep lung fibroblasts OAR-L1,and to explore a suitable transfection method for the cell line,the transfection efficiencies of polyethyleneimine(PEI),Lipofectamine^(TM) 2000 transfection reagent(Lipo 2000),Cytofect^(TM) fibroblast transfection kit(CF2)and lentivirus mediated cell transfection in the OAR-L1 cells were compared.The results showed that when OAR-L1 cells were transfected with fluorescent plasmids pLentiCMV-EGFP-Puro or pLentiCMV-mCherry-Puro,PEI-,Lipo 2000-and CF2-mediated transfection could be affected by the cell density and the amount of transfection reagents,besides the best transfection efficiency of each method was less than 30%.While the number of fluorescent cells obtained by lentivirus-mediated cell infection was not limited by these two factors,and was significantly higher than the former three methods.The recombinant virus solution could be stored at 4 or-80℃for at least 15 d without decline of the infection efficiency.To co-express two exogenous proteins in the OAR-L1 cells,mixing two packaged lentivirus in equal proportions followed by infection could achieve a higher co-transformation rate.The above results show that lentivirus infection is a cell transfection method that could achieve high expression of exogenous proteins in the OAR-L1 cells,and provide certain references for the selection of transfection methods for other difficult-to-transfect cells.

关 键 词：成纤维细胞OAR-L1 转染方法 慢病毒 绵羊 

分 类 号：S855.9[农业科学—临床兽医学]