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. 2010 May 12;5(5):e10606. doi: 10.1371/journal.pone.0010606

Figure 8. JCV early genes expressed from a CMV promoter induce replication of a JCV origin plasmid.

Figure 8

PHFG cells in 60 mm plates were co-transfected in duplicate with 400 ng of the JCV replication origin plasmid, pM1o, and 400 ng of one of the following expression vectors that encode JCV early genes under the control of the CMV promoter: pCMV:T+/t+/T′+ (lanes 2, 3; all five tumor proteins expressed), pCMV:T+/t−/T′+ (lanes 4, 5; TAg and 3 T′ proteins expressed) or pCMV:T+/t−/T′− (lanes 6, 7; TAg only expressed). The ability of the proteins produced by the second plasmid to drive replication of the origin plasmid in trans was tested. Duplicate, independent samples representing each DNA construct were extracted by the method of Hirt [39] on days 0, 3, 4 and 5 p.t. and analyzed using the Dpn1 assay as described in the legend to Figure 5. The marker (M) shown in lane 1 of each blot is 1 ng of linear pM1o (∼2400 bp), and the position of Dpn1-resistent replicating genomes is denoted by an arrow at days 3–5 p.t. Dpn1- and EcoRI-sensitive input DNAs are noted at the day 0 time point.