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. 2017 Jan 4;25(1):102–119. doi: 10.1016/j.ymthe.2016.10.005

Figure 2.

Figure 2

Characterization of the Functional Modules of the Synthetic Anti-inflammatory Gene Circuit

(A) Validation of the sensor construct (pAS48; PNF-κB2-PMIN-GV16) in HEK293T cells. Left: Dual luciferase test. Middle: confocal fluorescence microscopy (mCherry represents a seeding/transfection control). Right: model simulation of the stimulated and non-stimulated sensor-reporter pairs with increasing sensor amounts 24 hr after stimulation. (B) Validation of the amplifier construct (pAS53; UAS-PMIN-GV16) in HEK293T cells. Left: Dual luciferase test. Middle: confocal fluorescence microscopy (mCherry represents a seeding/transfection control). The amplifier autoactivates at higher amounts of transfected construct (cGV16− conditions and a confocal fluorescence image). Additionally, it amplifies an effect of constitutively expressed GV16 as observed in cGV16+ conditions and from FI calculated from the relative light units (RLU) for each amount of the transfected amplifier according the formula [cGV16+A+/cGV16+A−], where A− represents constitutive expression of GV16 from the pSGVP in the absence of the amplifier (A(0ng) in the RLU graph). Right: model simulation of the amplifier-reporter pairs with increasing amplifier amounts after 24 hr. The effect of cGV16 was examined by setting the particle number of GV16 at t = 0 to 1 × 106 for the cGV16+ simulation. (C) Functional circuit, composed of all the basic elements, except for the thresholder, tested in HEK293T cells. Left: dual luciferase test, reporter pAS51; TRE-UAS-PMIN-Luc. Right: model simulation and parameter scan. The graph and heat maps depict the level of the device activation in stimulated and non-stimulated setting with increasing sensor and amplifier amounts after 24 hr. The amount of each respective construct was increased from 0 to 10 ng, in 0.1-ng increments. Fold induction was calculated by dividing the effector particle number after 24 hr of stimulation with the corresponding effector particle number of non-stimulated circuit after 24 hr for each simulation. S, sensor; A, amplifier; FI, fold induction. Error bars indicate SD (n = 4).