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. 2009 Feb 11;83(8):3919–3929. doi: 10.1128/JVI.02466-08

FIG. 2.

FIG. 2.

The Rep5-TR5 interaction is unique among the fully characterized AAV serotypes. (A) Vector constructs used in this study. GFP expression was driven by a CMV promoter and simian virus 40 poly(A) element. The neomycin cassette included the thymidine kinase promoter and the bovine growth hormone poly(A) element. TR2s or TR5s flanked the vectors. pTR5-eGFP contained an additional 500 bp ahead of the 3′ TR. (B) Southern blot of Hirt DNA comparing the abilities of Rep2Cap2, Rep5Cap2, and Rep6Cap6 to replicate TR2s or TR5-flanked vector genomes. Hirt DNA was isolated 48 h after transfection. DpnI cuts only the input plasmid, not the newly replicated AAV genomes. The two major replicative forms of AAV are indicated (m, double-stranded monomer; d, double-stranded dimer). Higher-order replicative forms are also visible. +, present; −, absent. (C) Transduction of HEK 293 cells (transducible by Cap2) or CHO pgsD cells (transducible by Cap6 or Cap2) with crude lysate from cells harvested 48 h after transfection of an Ad helper plasmid only or triple transfection of an Ad helper plasmid, TR2 or TR5 GFP, and either Rep2Cap2, Rep5Cap2, or Rep6Cap6. The numbers shown correspond to the lane of the gel in Fig. 2B.