Efficient gene delivery into mammalian cells by baculovirus vector in vitro

Abstract

The recombinant baculovirus vector with EGFP reporter gene under the control of strong CAG promoter cassette was used for transduction of normal (HEK293) and tumor (HeLa) human cell lines. Dependence of transduction efficiency on the virus dose, time of virus incubation, temperature and cell type is shown. Transient reporter gene expression gradually diminished and was detected in single cells on the 15 th day after transduction.

Mouse embryonic fibroblasts expressing IFNβ or IL-21 inhibit proliferation of melanoma cells in vitro

“…It is known that BVs provide transient expression of transgenes in non-permissive mammalian cells in the absence of viral replication and a gradual loss of viral DNA upon cell division. As was shown earlier, the expression of GFP reporter protein may last for 30 days gradually decreasing, but stored in some single cells [28,30]. Using q-PCR, we showed that Ifnβ was expressed at a high level for 120 hours, substantially equal to that observed immediately after transduction (Fig.…”

“…The virus was concentrated by centrifugation at 100000 g. The titer of viruses preparations was 2-4×10 8 pfu/ml. Transduction was performed as desribed previously [28]. C57Fb cells were incubated with recombinant BVs at a concentration of 200 moi (multiplicity of infection = pfu per cell) for 4 hours at 28 °C in phosphate buffered saline (PBS), followed by addition of 1.5 ml DMEM medium and culturing for 16 hours at 37 °C.…”

The evidence of potential tumor suppressor properties of TAGLN in vitro

“…Plasmid pFastBac was used as a donor plasmid. According to the protocols, recombinant baculoviruses AcGFP were obtained under the CMV virus gene promoter for expression in mammalian cells with the Green fluorescent protein (GFP) gene embedded in the cassette under the CV40 promoter [20,21]. Recom bi nant baculovirus AcGFP-TAGLN contained both the TAGLN and the GFP gene in the cassette.…”

“…A monolayer culture of Sf21 insect cells was grown in TC-100 medium (Sigma) with the addition of 10 % FBS at 28 °C. Transfection of cells by recombinant baculoviruses was carried out according to the standard procedures [21].…”

“…Two prostate cancer cell lines (LNCaP, PC3), one conventionally normal prostate cell line (PNT2) and human embryonic kidney cell line HEK293 were used to study the potential tumor suppressor activity of the TAGLN in vitro. The transduction of these cell lines by recombinant baculoviruses with the TAGLN (AcGFP-TAGLN) and without it (AcGFP) was performed three times in three replicates [20,21]. All types of cell lines without baculoviruses under transduction procedures were performed as controls.…”