Investigation of optimal transduction conditions for baculovirus-mediated gene delivery into mammalian cells.
Although baculovirus-mediated gene delivery into mammalian cells has been documented in a wealth of the literature, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus is proposed for simple and efficient gene delivery into HeLa cells. We found that approximately 75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25 degrees C using Dulbecco's phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasts with previous protocols in which transduction occurs for 1 h at 37 degrees C using growth medium (e.g., DMEM) as the surrounding solution. Investigation of the physical parameters led to the findings 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 degrees C; 3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; and 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. 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 eukaryotic protein production on a larger scale.
Hsu
Ching-Suei
CS
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan 300.
Ho
Yi-Chen
YC
Wang
Kuei-Chun
KC
Hu
Yu-Chen
YC
D000818:Animals; D016367:Baculoviridae; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells / Q000821:virology*; D006801:Humans; D066298:In Vitro Techniques; D014161:Transduction, Genetic / Q000379:methods*; D014776:Virus Cultivation