Figure 3.

Establishment of the plasmid replicon after infection with the hybrid-vector system. (a) Experimental setup for long-term evaluation of the adenovirus/pEPito hybrid-vector system. U87 cells were coinfected with the FLPe-encoding virus HCAdV-FLPe and either HCAdV-pEPito-FRT² or HCAdV-pEPito-ΔS/MAR-FRT² at MOIs of 10, 30, and 100 for each virus. Cells were selected for 6 weeks using blasticidin, surviving cell colonies were stained with methylene blue and molecular analysis was performed. (b) Colony forming assay 6 weeks after blasticidin selection. Surviving colonies were stained with methylene blue. (c) Recombination PCR was performed with primers Recomb-fw and Recomb-rev on genomic DNA from U87 cells coinfected with HCAdV-pEPito-FRT² or HCAdV-pEPito-ΔS/MAR-FRT² and the FLPe-encoding virus HCAdV-FLPe. The expected PCR product was 530 bp in length. M, DNA ladder as marker. (d) Plasmid rescue 6 weeks after infection under 6 weeks of selection pressure. Genomic DNA was isolated and transformed into competent bacteria to grow single colonies of episomal plasmid replicons. The left panel shows a control digest of plasmid pEPito-FRT using the single-cutter BamHI serving as a positive control. The right panel shows exemplarily 5 rescued plasmids (R1-R5) of the group that received virus HCAdV-pEPito-FRT² and HCAdV-FLPe. M, DNA ladder as marker. (e) Overlapping PCR after 6 weeks of selection procedure. Principle of PCR (left panel): Two overlapping fragments I and II were amplified covering the complete pEPito-FRT plasmid. PCR was performed on genomic DNA from U87 cells coinfected with HCAdV-pEPito-FRT² and HCAdV-FLPe (right panel). Expected length of PCR product I amplified with primers Recomb-fw and BSD-IRES-rev-BamHI was 2.9 kb and the expected length of PCR product II amplified with primers Recomb-rev and BSD-IRES-fw-BglII is 3.9 kb.