Abstract
Although baculovirus-mediated gene delivery into mammalian cells has been reported in many literatures, previous studies were focused on the application of baculovirus as a gene therapy vector. Besides, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus was proposed for simple and efficient gene delivery into HeLa cell. We found that □75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25□C using Dulbecco’s phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasted to previous protocols in which transduction occurs for 1 h at 37□C using the growth medium (e.g. DMEM) as the surrounding solution. Investigation of the physical parameters led to the finding that (1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; (2) the half-life of baculovirus dramatically decreased at 37□C; (3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; (4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction; several factors (e.g. pH, ions, and glucose concentration) were tested to investigate the low efficiency caused by DMEM, but the reasons were not clear yet; (5) the transduction conditions we proposed was totally different from the usual culture conditions and the transduction conditions used by other groups. However, this protocol did not cause cytopathic effect but only transient effect on grow curve. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eucaryotic protein production in a larger scale.