Labeling of rat bone marrow mesenchymal stem cells with GFP infection by lentiviral vector

Objective: To determine the optimal condition for labeling rat bone marrow mesenchymal stem cells (MSCs) with green fluorescent protein (GFP) infection by lentiviral vector so as to establish a subgroup of MSCs which have a high level of GFP expression. Methods: MSCs were infected with GFP by lentiviral vector for 12 h or 24 h at different MOI (25, 50, 100, 200 and 400). The infection efficiency and fluorescence intensity were analyzed by inverted fluorescent microscopy and flow cytometry. The effect of infection at different MOI on proliferation of MSCs was evaluated by MTT. Based on those mentioned above, we could determine the optimal condition for infection. Then single cell clones of MSCs labeled with GFP under optimal condition were selected by using cloning rings. Results: The efficiency of infection for 24 h at MOI 100, 200 and 400 was 88.94%, 99.65% and 99.42%, respectively. The infection had no significant effect on the proliferation of MSCs infected at MOI 100 or 200, compared with MSCs without infection. However, those MSCs infected at MOI 400 proliferated slowly. The rate of GFP expression on single-cell clone of MSCs infected for 24 h at MOI 200 was 99.95%, and their fluorescence intensity was strong and uniform. Conclusion: Infection for 24 h at MOI 200 is optimal for labeling rat bone marrow MSCs with GFP by a lentiviral vector. A subgroup of MSCs which have a stably high level of GFP expression can be obtained by single cell cloning technique.