Application of NucleofectorTM technology in the transfection of rat nestin-positive cells derived from bone marrow with pancreatic duodenal homeobox-1 gene

Abstract：BACKGROUND: The direct gene transfer can enhance the regulation of the specific differentiation of bone marrow mesenchymal stem cells (BMSCs), thus, efficient gene transfer techniques are key to the use of BMSCs in tissue engineering.OBJECTIVE: To investigate the optimal transfection method for rat BMSCs termed Nucleofector TM technology, which provide a new method for high-efficient transfection of rat BMSCs in vitro.DISIGEN, TIME AND SETTING: Comparative observation, cell genetic engineering experiment was conducted between October and December 2007 at the Central Laboratory of the General Military Neurological Research Institute, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China.MATERIALS: BMSCs from clean Sprague-Dawley rats of 3-4-week-old, weighing 60-70 g.METHODS: BMSCs were isolated by density gradient centrifugation, nestin-positive cells were obtained by whole-bone-marrow culture followed by neural stem cell induction. BMSCs and nestin-positive cells were transfected with 1, 5, 10 μ g recombinant pla.smid pEGFP-C1 using A33 and A31 program of NucleofectorTM technology, transfected cells were cultured in 10% and 20% fetal bovine serum medium, respectively.MAIN OUTCOME MEASURES: Cells expressed green fluorescence and transfection rote counted under LeicaDMIRE fluorescence microscope; cell viability determined on the basis of trypan blue dye exclusion; the expression of pancreatic duodenalhomeobox- 1 (PDX- 1) gene in the transfected cells analyzed by reverse transcription polymerase chain reaction.RESULTS: The A33 program of Nucleofector TM technology was more efficient in the transfection of neural stem cells than BMSCs, it was also more efficient than A31.The 24-hour transfection rate and survival rate of transfected cells cultured in the medium with 20% fetal bovine serum were markedly higher than those cultured in the medium with 10% fetal bovine serum (P< 0.05). In reverse transcription polymerase chain reaction detection, mRNA of PDX-1 gene was shown in nestin-positive cells transfected with recombinant plasmid, and not found in those without recombinant plasmid transfection.CONCLUSION: We obtained higher transfection rate and survival rate when we transfected nestin-positive cells derived from bone marrow with 5 μ g DNA using A33 program of Nucleofector TM technology and then cultured the transfected cells in the medium with 20% fetal bovine serum.