3种转染试剂对牛原代骨骼肌卫星细胞转染条件的优化

doi:10.16431/j.cnki.1671-7236.2019.01.008

Abstract

Abstract:

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 1.0 μL Lipofectamine 3000, 1.5 μL 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 1.0 and 1.5 μL 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.

Key words: primary bovine skeletal muscle satellite cells; transfection; Lipofectamine 3000; X-tremeGENE HP; FuGENE HD

CLC Number:

CHEN Mingming, LI Yan, ZHANG Xiaojuan, ZHANG Linlin, LI Xin, DING Xiangbin, LIU Xinfeng, GUO Hong. Optimization of Transfection Conditions About Three Kinds of Transfection Reagents on Primary Bovine Skeletal Muscle Satellite Cells[J]. , 2019, 46(1): 62-71.

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

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