JOURNAL ARTICLE

[Construction of lentiviral vector containing homo sapiens forkhead box C2 gene and its expression in bone marrow mesenchymal stem cells of rabbits]

Wulin You, Kunzheng Wang, Dapeng Duan, Chunsheng Wang, Lihong Fan, Ruiyu Liu
Chinese Journal of Reparative and Reconstructive Surgery 2013, 27 (5): 535-40
23879088

OBJECTIVE: To construct the lentiviral vector containing homo sapiens forkhead box C2 (Foxc2) gene and

METHODS: Human Foxc2 gene coding to detect its expression in bone marrow mesenchymal stem cells (BMSCs) of rabbits. region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein (GFP) to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelperl.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection (MOI) was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule.

RESULTS: The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 x 10(8) TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5% +/-4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm.

CONCLUSION: Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cellls may be applied for gene therapy of avascular necrosis of the femoral head.

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