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. 2023 May 10;11:1168330. doi: 10.3389/fbioe.2023.1168330

FIGURE 5.

FIGURE 5

Validation of the ON–OFF genetic switch in vitro and in vivo. (A) Schematic representation of the OFF and ON systems. For an OFF system, in the absence of dox, the activator binds upstream of a minimal promoter and activates the gene of interest (GOI). Upon addition of doxycycline, the activator dissociates and the gene does not express. On the contrary, in the case of the ON system, gene is not expressed in the absence of an inducer. Upon induction with doxycycline, the activator binds to a minimal promoter and activates GOI. (B) Time-dependent deactivation or (D) activation of inducible systems. Data are presented as mean ± SEM (B, n = 16; D, n = 8), representative of three independent experiments. (C,E) Responsiveness of systems to doxycycline (0.01, 0.05, 0.1, 0.5, 1, 5, and 10 μg/mL). Data are presented as mean ± SEM (n = at least 3), representative of three independent experiments is shown. (F) Hollow fibers are filled with OFF or ON stable cell lines and photographed before induction with doxycycline (1 μg/mL; time 0) and after 24 h using an IVIS system. (G) Hollow fibers filled with OFF or ON stable cell lines are implanted subcutaneously into BALB/c mice and visualized at times 0 and 24 h after intraperitoneal doxycycline induction (20 μg/g) using the IVIS system.