摘要
试验旨在探究利用携带标记基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的腺病毒载体转染入水貂耳缘成纤维细胞的可行性及感染效率。首先利用组织贴壁法分离培养水貂原代耳缘成纤维细胞并进行传代,然后包装病毒Ad-EGFP并使用TCID 50法进行滴度检测,设置两组不同浓度梯度的病毒液感染水貂耳缘成纤维细胞,感染复数(MOI)值分别为0/100/300/500/700/900和0/10/30/50/70/90,通过在显微镜下观察阳性细胞占总细胞数的比值估计转染效率(%)。结果表明,组织贴壁法培养水貂耳皮肤组织11 d可长满原代细胞,传代细胞在3~4 d融合度可达80%左右,包装Ad-EGFP病毒滴度为1.99×1011 PFU/mL,用其感染水貂成纤维细胞最佳MOI值为30,瞬时转染效率可达90%以上。综上,利用腺病毒载体转染水貂成纤维细胞具有可行性,是一种高效的转染方式,为后续水貂基因组编辑前期验证试验奠定基础。
The objective of this study was to investigate the feasibility and efficiency of transfection of adenovirus vector with enhanced green fluorescent protein(EGFP)into fibroblasts in the ear margin of mink.Firstly,ear fibroblasts of mink were isolated and cultured by tissue adherent method,and then the virus Ad-EGFP was packaged and the titer was detected by TCID 50 method.Two groups of different concentration gradients were used to infect mink fibroblasts,multiplicity of infection(MOI):0/100/300/500/700/900 and 0/10/30/50/70/90.Transfection efficiency(%)was estimated by looking at the ratio of positive cells to the total number of cells under a microscope.The results showed that primary cells could be grown in the ear tissue of mink cultured by tissue adherent method for 11 d,and the fusion degree of passage cells could reach 80%in 3-4 d.The titer of the packaged Ad-EGFP virus was 1.99×1011 PFU/mL,the optimal MOI value of the infected mink fibroblasts was 30,and the instantaneous transfection efficiency was more than 90%.In conclusion,it was feasible to transfection mink fibroblasts with adenovirus vectors,which was an efficient transfection method and provided a basis for the preliminary validation test of mink genome editing.
作者
李文
韩玉萍
赵向远
范冰峰
李晓霞
刁云飞
杨镒峰
许保增
LI Wen;HAN Yuping;ZHAO Xiangyuan;FAN Bingfeng;LI Xiaoxia;DIAO Yunfei;YANG Yifeng;XU Baozeng(State Key Laboratory for Molecular Biology of Special Economic Animals,Institute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130112,China)
出处
《中国畜牧兽医》
CAS
北大核心
2020年第1期37-43,共7页
China Animal Husbandry & Veterinary Medicine
基金
吉林省国际科技合作项目“水貂基因组编辑配套技术体系的研究与集成”(20170414049GH)
关键词
水貂
腺病毒转染
成纤维细胞
细胞培养
American mink
adenovirus transfection
fibroblast
cell culture
