Construction of Lentiviral Expression Vector Containing Mouse Smad4 Gene and Its Expression in 293T Cells

Objective To construct a lentivirus expression vector containing the mouse smad4 gene and EGFP gene for possible application in recombinant lentivirus-mediated inner ear gene transfer and gene therapy for deafness. Methods The eukaryotic expression plasmid pLenti6.3-Smad4-IRES2-EGFP was constructed and identified by gene recombination, restriction enzyme digestion and gene sequencing methods. The 293T cells were transfected with the pLenti6.3-Smad4-IRES2-EGFP plasmid by the lipofectamine-mediated transfection method EGFP gene expression was examined under a fluorescence microscope. Mouse Smad4 mRNA expression was detected by real-time PCR. Results The PCR and sequencing analysis confirmed that the mouse smad4 gene sequence was consistent with the reference in the gene bank. After 293T cells were transfected with the pLenti6.3-Smad4-IRES2-EGFP plasmid, the green fluorescent protein was visible. After 293T cells were infected with lentivirus, Smad4 gene mRNA expression increased by 66,427 times, reaching to a titer of 2.5 ×108TU/ml. Conclusions The recombinant lentivirus expression vector for mouse smad4 gene can be constructed with successful vector expression in 293T cells.