Expression of pIRES2-AcGFP1-CD eukaryotic expression plasmid in bone marrow mesenchymal stem cells

Background: Bone marrow mesenchymal stem cells (BMSCs) are easy to isolate and amplify in vitro, and easy for transferring and expressing of exogenous gene. BMSCs are ideal therapeutic cells and target cells for gene therapy. Objective: To investigate transfection and expression of liposome-mediated cytosine deaminase gene in rabbit BMSCs. Design, time and setting: The single sample cell gene engineering experiment was performed at the Dalian Research & Development Center for Stem Cells and Tissue Engineering, Dalian University of Technology from March 2006 to April 2007. Materials: New Zealand big-eared rabbits aged 5 months were used for isolation and culture of BMSCs. Methods: BMSCs were harvested by density gradient centrifugation. The cytosine deaminase gene was obtained from E.coli JM109 genome DNA by polymerase chain reaction. The fragment was cloned into pMD19-T vector. Restriction enzyme BamHI/XhoI digestion analysis and DNA sequence analysis showed that CD gene was identical with the published sequence. Constructing the pIRES2-AcGFP1-CD plasmid and identified by Restriction enzyme BamHI/XhoI digestion analysis. Lipofectamine 2000-mediated cytosine deaminase gene was used to transfect BMSCs. Main outcome measure: Expression of green fluorescent protein was identified by inverted fluorescent microscope. Results: We cloned cytosine deaminase gene and the results from gene sequencing were the same as Genbank. Eukaryotic expression plasmid was constructed after cytosine deaminase gene was cloned on pIRES2-AcGFP1 plasmid. Cytosine deaminase gene was transfected on BMSCs by liposome-mediated method. Twenty-four hours later, expression of green fluorescent protein was measured with an inverted fluorescent microscope. Conclusion: pIRES2-AcGFP1-CD eukaryotic expression vector has been successfully transfected into BMSCs, which indicates that BMSCs can be an ideal vector for cytosine deaminase gene therapy.