3种转染试剂对牛原代骨骼肌卫星细胞转染条件的优化

Optimization of Transfection Conditions About Three Kinds of Transfection Reagents on Primary Bovine Skeletal Muscle Satellite Cells

为了选择适合牛原代骨骼肌卫星细胞的转染试剂与条件,本研究以带有绿色荧光蛋白(EGFP)的质粒pcDNA3.1-EGFP和pEGFP-C1作为外源基因,分别用Lipofectamine 3000、X-tremeGENE HP和FuGENE HD转染牛原代骨骼肌卫星细胞,通过荧光显微镜观察、实时荧光定量PCR及Hoechst 33342染色评价转染效率。结果表明,对于同一时期、同一转染试剂,pcDNA3.1-EGFP转染效果优于pEGFP-C1;不同转染试剂之间,Lipofectamine 3000和X-tremeGENE HP转染效果优于FuGENE HD。Lipofectamine 3000转染后分化72h肌管最明显,FuGENE HD次之,X-tremeGENE HP转染后细胞死亡较多。采用1.0μL Lipofectamine 3000、1.5μL Lipofectamine 3000和X-tremeGENE HP转染细胞后EGFP表达量极显著高于FuGENE HD (P<0.01),但这三者之间差异不显著(P>0.05)。细胞转染效率与EGFP定量结果基本一致,1.0、1.5μL Lipofectamine 3000转染效率较高,分别达26.07%和24.77%,极显著高于FuGENE HD(5.59%)和X-tremeGENE HP(10.87%)的转染效率(P<0.01),但二者之间无显著差异(P>0.05);X-tremeGENE HP的转染效率极显著高于FuGENE HD (P<0.01)。综合考虑转染试剂的用量、细胞毒性及经济效益,本试验初步认为牛原代骨骼肌卫星细胞转染宜采用Lipofectamine 3000,其与质粒DNA的比例为1∶1时转染效率最高。本试验结果可为原代细胞的转染提供重要的参考价值。

To select a suitable transfection reagent and condition for primary bovine skeleltal muscle satellite cells,the pcDNA3.1-EGFP and pEGFP-C1 plasmids with green fluorescent protein(EGFP)were used as exogenous gene to transfect primary bovine skeletal muscle satellite cells by Lipofectamine 3000,X-tremeGENE HP and FuGENE HD in this study.Then the transfection efficiency was evaluated by fluorescence microscopy,Real-time quantitative PCR and Hoechst 33342 staining.The results showed that transfection efficiency of pcDNA3.1-EGFP was better than pEGFP-C1 for the same period and transfection reagent.Among different transfection reagents,the effect of transfections in Lipofectamine 3000 and X-tremeGENE HP were superior to FuGENE HD.Myotubes were most obvious with Lipofectamine 3000 transfection after differentiation 72 h,followed by FuGENE HD,X-tremeGENE HP transfection leaded to more cell death.The expression of EGFP was extremely significantly higher than FuGENE HD after transfection with 1.0μL Lipofectamine 3000,1.5μL Lipofectamine 3000 and X-tremeGENE HP(P<0.01),but there was no significant difference in three groups(P>0.05).The cell transfection efficiency was basically consistent with the result of EGFP quantification,the transfection efficiency of 1.0and 1.5μL Lipofectamine 3000 was extremely significantly higher than FuGENE HD(5.59%)and X-tremeGENE HP(10.87%)(P<0.01),respectively up to 26.07% and 24.77% with no significant difference(P>0.05).The transfection efficiency of X-tremeGENE HP was also extremely significantly higher than FuGENE HD(P<0.01).Comprehensive consideration the amount of transfection reagents,cytotoxicity and economic benefits,this study preliminarily assumed that the transfection of primary bovine skeletal muscle satellite cells should adopt Lipofectamine 3000 transfection reagent,and the transfection efficiency was the highest when the ratio of Lipofectamine 3000 to plasmid DNA was 1∶1.This experiment could provide important references for transfection of primary cells myogenic.