Abstract
Baculovirus transduction of cultured mammalian cells is typically performed by incubating the cells with virus using culture medium (e.g. DMEM) as the surrounding solution. However, we previously uncovered that DMEM hinders the baculovirus-mediated gene transfer. In this study, we demonstrated that the poorer transduction by using DMEM as the surrounding solution is independent of the promoter. Examination of the medium constituents group by group revealed that the balanced salt solution suppresses the baculovirus transduction. By omitting individual salt species in the balanced salt solution, we surprisingly uncovered that NaHCO3, a common buffering agent, exerts the inhibitory effects in a concentration-dependent manner. Intriguingly, NaHCO3 did not debilitate the baculovirus, nor did it inhibit virus binding to the cells as revealed by immunofluorescence/confocal microscopy. Instead, NaHCO3 inhibited baculovirus transduction by reducing the intracellular virus number as demonstrated by quantitative real-time PCR. To our best knowledge, this is the first report unraveling the significance of NaHCO3 in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO3 from the medium during the transduction period. Furthermore, we explored the feasibility of employing baculovirus in muscle-based gene therapy in order to augment the short baculovirus-mediated expression. We demonstrated that baculovirus transduces myoblast cells efficiently and the duration of baculovirus-mediated expression is prolonged to at least 60 days after the myoblast cells differentiate into multi-nuclei myotubes. The prolonged expression also paralleled the increased cellular expression of MyoD and myogenin as demonstrated by RT-PCR, and resulted from slower degradation of viral DNA and persistent presence of the transgene mRNA, as confirmed by quantitative real-time PCR. These data collectively suggested a correlation between the prolonged expression and myogenic differentiation which resulted in extended existence of viral DNA and transgene mRNA, implicating the tremendous potential of baculovirus vector in muscle-based gene therapy.