Investigation of optimal transduction conditions for baculovirus‐mediated gene delivery into mammalian cells

Abstract

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.

Baculovirus-Mediated Gene Delivery into Mammalian Cells Does Not Alter Their Transcriptional and Differentiating Potential but Is Accompanied by Early Viral Gene Expression

“…2D). As has been previously reported for AcNPV-based vectors (33,36), maximal efficiency of transduction was achieved by using Dulbecco's PBS as the transduction medium, with higher efficiencies of transduction being achieved at 28°C (data not shown).…”

Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy

“…Due to the integration, the hybrid SB-baculovirus vectors allowed for prolonged expression of EGFP ( Figure 2a) and hEA (Figure 3b), whose durations considerably outlasted those of the conventional baculovirus vectors (typically o7 days in dividing cells 33 ). The Bac-SB-T2hEA/w-mediated hEA expression was robust initially (for example, at 2 d.p.t.)…”

Generation of lentivirus vectors using recombinant baculoviruses

“…This is in line with the fact that the transduction medium affects baculovirusmediated gene expression in vertebrate cells. 34,35 To confirm the functionality of the generated lentiviruses, HeLa cells were transduced and sustained GFP expression was observed for 6 weeks. On the contrary, with the control baculovirus the GFP expression was lost in 17 days.…”