Abstract:

BACKGROUND: The use of mesenchymal stem cells or therapeutic factor-containing stem cells is a promising therapeutic method to selectively kill tumor cells.
OBJECTIVE: To establish rat bone marrow mesenchymal stem cell lines stably transfecting enhanced green fluorescent protein.
METHODS: The lentiviral plasmid pVector-EGFP with the packaging plasmid pHelper 1 and the envelop plasmid pHelper 2 were co-transfected into 293T cells. The lentiviral titer was detected by real-time, fluorescence-based quantitative PCR. SD rat bone marrow mesenchymal stem cells during logarithmic phase were seeded in 24-well plates and exposed to lentiviral vector coding for EGFP reporter gene at multiplicity of infection (MOI) of 0, 5, 10, 15 and 20, respectively. EGFP expression and transfection efficiency were determined 72 hours later.
RESULTS AND CONCLUSION: The lentiviral vector system carrying EGFP was successfully tranfected into 293T cells with a lentivirual titer of 1×10⁸ TU/mL. At 2-3 days after rat bone marrow mesenchymal stem cells were transfected with packaged lentiviral vector coding for EGFP reporter gene, EGFP expression was detected in each well. MOI values ranged from 0 to 10. EGFP-expressing cells were gradually increased (P < 0.05). Over 70% cells were EGFP-positive at 3 days after transfection by lentiviral vector at MOI of 10. A significant dose-response was observed with increasing lentiviral titer for MOI 0 to 10 (P < 0.05), whereas the ratio of transfection decreased with increasing lentiviral liter for MOI 10 to 20. These findings suggest that lentivirus is an ideal vector for gene transduction and it can transduce an exogenous gene into rat bone marrow mesenchymal stem cells with over 70% efficency at the MOI of 10 to establish rat bone marrow mesenchymal stem cell lines stably transfecting EGFP.