Transfection by the green fluorescent protein gene and cloning culture in mesenchymal stem cells derived from rat bone marrow

Objective To establish a cell line drived from rat bone marrow mesenchymal stem cells (MSCs) which stably,high-level express green fluorescent protein (GFP) and pass on its cultivation continuously.Methods The transfected rat MSCs were obtained with pGFP-N1 vector containing the green fluorescent protein (GFP) gene by electroporation in various conditions.The MSCs expressing GFP- were detected by fluorescent microscopy and flow cytometer through G418 selecting and cloned culture in limiting dilution.Results In the optimum electroporation condition:voltage at 260 volt,capacitance at 1050μF,transfection of rat MSCs with green fluorescent protein (GFP) gene displayed a stable and long-term GFP expression in vitro,and untransfected rat MSCs gradually died in G418 selecting culture.Bright green fluorescence of the transfected cells could be observed under fluorescent microscopy after 24h of transfection and displayed confluence after one month. The gene transfection efficiency measured by GFP fluorescence was 50%～60%. In culture of two months, the transfected cells could express high-intensive and stable GFP (for 10 passages),but the green fluorescence was obviously weakened after 10 passages.Conclusions The GFP gene could be effectively expressed in rat MSCs,the transfected cells showed stable and long-term GFP expression in vitro.These results illustrated transfected MSCs expressing GFP could be used to study on differentiation of MSCs and their potential utility as cellular vehicles for systemic gene delivery.