Figure 2.

Optimization of infection conditions and basic features of the hybrid-vector system. (a) U87 cells were infected with HCAdV-pEPito-FRT² and HCAdV-pEPito-ΔS/MAR-FRT² at MOI of 200 and either coinfected with the stuffer virus HCAdV-hFIX (see Figure 1e) at MOI of 200 or the FLPe-encoding virus HCAdV-FLPe at MOI of 200, respectively (total MOI of 400 for each coinfection experiment). Seventy-two hours after infection percentages of eGFP-expressing U87 cells were analyzed by flow cytometry (top panel). Mean values of three independent experiments (±SD) are shown; *P < 0.05. To prove excision, a PCR was performed with primers Recomb-fw and Recomb-rev on genomic DNA from U87 cells 72 hours after infection (bottom panel). The expected PCR product is 530 bp in length. For details of the PCR setup, please also refer to Supplementary Figure S5. (b) Plasmid rescue of excised plasmid replicons pEPito-FRT and pEPito-ΔS/MAR-FRT. Total genomic DNA was isolated and recircularized pEPito-FRT and pEPito-ΔS/MAR-FRT were rescued from U87 cells 72 hours after infection. Rescued plasmids were analyzed by restriction enzyme digest using the restriction enzyme XhoI. Exemplary XhoI-digests of two rescued pEPito-FRT and two pEPito-ΔS/MAR-FRT plasmids are shown. M, DNA ladder as marker; MOI, multiplicity of infections.