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

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Application of the aptazyme for regulation of gene expression by an adenoviral vector (AdV) in a replication-competent and replication-deficient model. (A) Schematic outline of a replication-deficient, first generation AdV. The essential E1 genes were replaced (ΔE1) by the shown firefly luciferase transcription units. Nomenclature for aptazyme and control constructs is as in Figure 2A. LITR, left inverted terminal repeat; Ψ, packaging signal; RITR, right inverted terminal repeat; ΔE3, E3 region deleted (not required for virus replication); other viral genes and elements are not shown (dashed lines). (B–F) Cells were transduced with indicated AdVs, and gene expression was measured at 48 h (B–F) or at indicated time points (D) post transduction. Shown are relative luciferase activities for AdVs 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 transductions, 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 *** over bars (P < 0.001). (B) Transduction of E1-complementing HEK293 cells with indicated AdVs (5 TCID₅₀/cell). Dashed line, reference gene activity level for calculation of aptazyme performance. §, absolute reporter gene expression for HHR 5′3′ was <1% compared with inHHR 5′3′. Theophylline-dependent regulation of gene expression was significantly superior for 5′3′ compared with both 5′ and 3′ as indicated by *** (P < 0.001). (C) Dose-dependent regulation of transgene expression by theophylline after transduction of HEK293 cells with indicated AdVs (5 TCID₅₀/cell). (D) Regulation of transgene expression at 10, 24 and 48 h after transduction of HEK293 cells with the indicated AdVs (5 TCID₅₀/cell). *(P < 0.05) and **(P < 0.01) as calculated above; c, P < 0.05 for relative reporter gene expression in comparison with ctrl only. (E) Transduction of HEK293 cells with indicated AdVs (5 TCID₅₀/cell). Theophylline was added either after transduction as before (standard), was added after transduction and removed at 24 h, or was added at 24 h post transduction. (F) Transduction of HEK293 (5 TCID₅₀/cell), HeLa (50 TCID₅₀/cell), SK-MEL-28 (100 TCID₅₀/cell) or A549 (50 TCID₅₀/cell) cells with indicated AdVs.