FV supernatants containing recombinant viral particles were generated essentially as described previously [51 (link),52 (link)]. Briefly, FV supernatants were produced by cotransfection of 293T cells with transfer vector (puc2MD9 or pMD11), Env- (pczHFVenvEM002), Pol - (pcziPol), and Gag packaging plasmid (pcoPG4 or PG mutants thereof as indicated) at a ratio of 4:4:4:1 using polyethyleneimine (PEI) or Polyfect transfection reagents. At 24 h posttransfection, sodium butyrate (final concentration, 10 mM) was added to the growth medium for 8 h. Subsequently, the medium was replaced, and viral supernatants were harvested an additional 16 h later. Lentiviral supernatants were generated by cotransfection of transfer vector (p6NST90), Gag/Pol packaging plasmid (pCD/NL-BH), and an Env packaging plasmid (pczVSV-G or pczPE01) at a ratio of 1:1:1 and harvested as described above.
Transductions of recombinant EGFP expressing PFV vector particles containing various PFV Gag proteins were performed by infection of 2 × 104 HT1080 cells, plated 24 h in advance in 12-well plates. For the infection 1 ml of the viral supernatant or dilutions thereof were incubated with the target cells for 4 to 6 h. The percentage of EGFP-positive cells was determined by fluorescence-activated cell sorter (FACS) analysis 72 h after infection. All transduction experiments were performed three times, and in each independent experiment the values obtained with the wild-type construct pcoPG4 were arbitrarily set to 100%. Analysis of tissue tropism of different PFV vector particles or HIV-1 vector pseudotypes was performed on various target cell lines. Adherent target cells (HT1080, HeLa, mouseL, Sog9, Pac2), which were plated one day in advance at a density of 2 × 104 cells in 12-well plates, were infected with one ml of viral cell culture supernatant or dilutions thereof. Target cells growing in suspension (Jurkat, G1E-ER4) were infected by resuspending 1 × 105 target cells in one ml of viral cell culture supernatant or dilutions thereof. Afterwards they were transferred into a 6-well plate and either incubated at 37°C, 5% CO2 in a humidified incubator or centrifuged for 1 h at 2000 rpm (30°C) before the incubation step. Sixteen hours later the viral cell culture supernatant was replaced for both types of target cells by fresh media and the transduction efficiency was determined by flow cytometry 72 - 96 h after infection as described above.
Transductions of recombinant EGFP expressing PFV vector particles containing various PFV Gag proteins were performed by infection of 2 × 104 HT1080 cells, plated 24 h in advance in 12-well plates. For the infection 1 ml of the viral supernatant or dilutions thereof were incubated with the target cells for 4 to 6 h. The percentage of EGFP-positive cells was determined by fluorescence-activated cell sorter (FACS) analysis 72 h after infection. All transduction experiments were performed three times, and in each independent experiment the values obtained with the wild-type construct pcoPG4 were arbitrarily set to 100%. Analysis of tissue tropism of different PFV vector particles or HIV-1 vector pseudotypes was performed on various target cell lines. Adherent target cells (HT1080, HeLa, mouseL, Sog9, Pac2), which were plated one day in advance at a density of 2 × 104 cells in 12-well plates, were infected with one ml of viral cell culture supernatant or dilutions thereof. Target cells growing in suspension (Jurkat, G1E-ER4) were infected by resuspending 1 × 105 target cells in one ml of viral cell culture supernatant or dilutions thereof. Afterwards they were transferred into a 6-well plate and either incubated at 37°C, 5% CO2 in a humidified incubator or centrifuged for 1 h at 2000 rpm (30°C) before the incubation step. Sixteen hours later the viral cell culture supernatant was replaced for both types of target cells by fresh media and the transduction efficiency was determined by flow cytometry 72 - 96 h after infection as described above.
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