Objective:To construct green fluorescent protein(GFP)retroviral vector(pLgXSN),and to investigate the expression of GFP in primary rat myoblast.Methods:GFP cDNA was subcloned into the plasmid pLgXSN,and the recombinant vector was transfected into packaging cell PT67.G418 was used to select positive colony.Myoblasts were infected by a high-titer viral supernatant.The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis.Confocal microscopy and flow cytometry were used to detect the expression of GFP.Results:The GFP cDNA sequence was identical to that of GenBank.Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully.The titer of the packaged recombinant retrovirus was 1 × 106 cfu/ml.Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection.The transfection rate was 33%.The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks.Conclusion:GFP gene could be effectively and stably expressed in myoblast,which suggests that GFP could act as a marker for studies on myoblast.
Shuling RongYongxin LuYuhua LiaoXiaolin WangXiaoqing LiJiahua ZhangYanli He
