Figure 4.

Analysis of persistence of transgene expression after delivery of the hybrid-vector system. eGFP expression was measured 72 hours after infection and 6 weeks after blasticidin selection. U87 cells were coinfected with the FLPe-encoding virus HCAdV-FLPe and either HCAdV-pEPito-FRT² or HCAdV-pEPito-ΔS/MAR-FRT² at MOI of 30 for each virus. Cells were selected for 6 weeks using blasticidin and surviving cell colonies were analyzed. (a) Flow cytometer analysis measuring percentages of eGFP-expressing U87 cells before and after treatment with blasticidin for 6 weeks (left panel). The right panel shows the mean intensity of eGFP expression of the same experiment. Exemplary results of one experiment are shown. (b) Original histograms of exemplary samples after flow cytometry showing eGFP-expressing U87 cells 72 hours (top panel) and 6 weeks (bottom panel) after infection and selection pressure using blasticidin. (c) Fluorescence microscopy 6 weeks after infection and selection pressure using blasticidin. Groups which received the adenoviral vectors HCAdV-pEPito-FRT² or HCAdV-pEPito-δS/MAR-FRT² along with the FLPe-encoding virus were analyzed. MOI, multiplicity of infections.