Transient transfection
Recombinant AAV6 particles expressing GFP driven by the CAG promoter
were produced via triple transient transfection of HEK293SF cells as
previously described [56]. In brief, the transgene plasmid
pAAV-CAG-GFP, the plasmid pRep2Cap6, and the helper plasmid pAdDeltaF6
were co-transfected into HEK293SF cells in a ratio of 1:1:1 after
complexation with 25-KDa linear polyethylenimine (PEI, Polysciences,
USA) at a ratio of 1:2 (DNA:PEI). Plasmid DNA and PEI were diluted
separately in HyCell TransFx-H in a volume corresponding to 2.5% of the
volume of cells to be transfected. For experiments where the DNA was
delivered on a volumetric basis, 1 µg of plasmid was added per
millilitre of cell culture. For delivering DNA on a cell basis, 1 µg of
plasmid was added per million viable cells. Diluted DNA and PEI were
mixed and incubated at room temperature for 15 minutes. The DNA-PEI
cocktail was then added to the cells.