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. 2012 Aug 9;40(21):e167. doi: 10.1093/nar/gks734

Figure 2.

Figure 2.

Characterization of different aptazyme insertion sites, and combinations thereof, using plasmid transfection. (A) Schematic representation of the analyzed aptazyme reporter shuttle plasmids (pS), which were also subsequently used for cloning of AdV genomes. Insertion sites for the aptazyme P1-F5 were within the 5′- and/or 3′-UTR of the transgene (orange box) driven by a SV40 promoter (green arrow). Brown box, synthetic polyadenylation signal; dark brown box, polyadenylation signal (pA). Nomenclature: 5′-UTR insertion distant (5') or proximal (5*) to the start codon, 3′-UTR insertion (3'), or in combinations thereof (5′5*, 5′3′ and 5′5*3'); ctrl, no aptazyme insertion. (B–D) HEK293 cells were transfected with the indicated reporter plasmids, harvested 48 h post transfection and gene activity/expression was measured. Shown are relative reporter gene activities or relative CCL5 expression for plasmids in the presence of theophylline (3 mM, except in C) in % of expression levels in the absence of theophylline (set as 100%, dotted line) of a representative experiment. Columns/symbols show mean values of triplicate transfections, error bars show SD. Significance for both theophylline-dependent down-regulation of gene expression for individual constructs and relative reporter gene expression in comparison to ctrl is indicated with ***(P < 0.001). (B) Regulation of gene expression for plasmids with firefly luciferase reporter gene by theophylline (3 mM). Dashed line, reference gene activity level for calculation of aptazyme performance. Controls are: in5′3′, inactive P1-F5 aptazyme in 5′- and 3′-UTR; HHR 5′3′, hammerhead ribozyme in 5′- and 3′-UTR; inHHR 5′3′; inactive HHR in 5′- and 3′-UTR. §, absolute reporter gene expression for HHR 5′3′ was <1% compared with inHHR 5′3′. (C) Dose-dependent regulation of the 5′3′ firefly luciferase construct and of the controls in5′3′ and ctrl by theophylline. (D) Regulation of gene expression for constructs 5′, 3′ and 5′3′ containing different reporter genes (Luc, firefly luciferase; Ren, renilla luciferase; LacZ, β-galactosidase) or the gene for human chemokine CCL5 by theophylline (3 mM).