摘要
C2C12细胞是常用于研究的肌细胞,广泛适用于肌肉再生、肌肉疾病、糖脂代谢以及药物研究实验。由于肌细胞结构较特殊,基因转染不容易成功。本文介绍脂质体介导的肌细胞瞬时转基因技术,国内外未见详细报道。我们实验室经优化细胞密度、质粒DNA纯度以及转染最佳时间等条件,并改变底物和稀释液的成份,将细胞分成几组:1底物Opti-MEM+稀释物DMEM组;2底物Opti-MEM+稀释物Opti-MEM组;3底物无血清DMEM+稀释物无血清DMEM组;4底物无血清DMEM+稀释物Opti-MEM组;5底物含10%FBS的DMEM+稀释物DMEM组进行转染。24 h后荧光倒置显微镜观察各组荧光表达情况,48 h Western Blot检测GFP蛋白表达。结果 C2C12细胞瞬时转染的细胞数量和荧光强度达到理想效果。其中第3组GFP蛋白表达水平明显高于第5组(p<0.05)。这是一种方便有效的脂质体介导C2C12细胞转基因技术。
C2C12 cells are often used to study the muscle cells, and are applied widely in muscular regeneration,muscular disease, glycolipids metabolism and drug experiment. Because the structure of the muscle cell is special,gene transfection is not easy to succeed. This article introduces the instantaneous transgenic technology in muscle cells mediated by liposome, but no detailed reports at home and abroad. After the optimized conditions in the cell density, the concentration of plasmid DNA and the best time of the transfection, and change the ingredients of substrate and diluent, our lab divided the C2C12 cells into several groups: 1 the substrate Opti-MEM+the dilution DMEM; 2 the substrate Opti-MEM+the dilution Opti-MEM; 3 the serum-free substrate DMEM+the serum-free dilution DMEM; 4 the serum-free substrate DMEM+the dilution Opti-MEM; 5 the substrate DMEM containing10% FBS+the dilution DMEM by transfection. After 24 hours, it could observe the expression of fluorescence under the fluorescence microscope in each group, and western blot detection of GFP protein expression in 48 hours.From the results, C2C12 cells number and fluorescence intensity of transient transfection achieved the ideal effect.Of those, GFP protein expression in the third group was significantly higher than that of fifth group(p〈0.05). This is a convenient and effective method of liposome mediated C2C12 gene transformation.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2016年第4期783-787,共5页
Genomics and Applied Biology
基金
国家自然科学基金(No.81160102)资助