Fig. 1. Optimization of IQ-Switch for toxicity and leakiness, while retaining transgene-stimulating activity.

a Alignment of driver and effector constructs tested for the optimization of IQ-Switch. b Schematic drawing of an executing mechanism of IQ-Switch. The chemical structure of tebufenozide is depicted on the right. c Measurement of luciferase reporter activity after transfection of an individual driver plasmid, indicated by number, together with the same effector containing 5xQUAS elements. After 24 h, the transfected HEK293 cells were exposed to 10 µM of Teb for another 24 h before measurement. The luciferase reporter activity responding to Teb is indicated in azure; the basal leakiness of the effector by a specific inducer plasmid is visualized in reddish brown. d The combination of driver ‘3’ in (a) with the effector in HEK293 cells showed responsiveness in a Teb dosage-dependent manner. Error bars stand for the standard deviation. e Reversibility of IQ-Switch. The offspring of the genetic cross between Tg(ubb:QFDBD-2xAD*-VP16*-EcR) and Tg(5xQUAS:ZsGreen-P2A-lamin A^Δ37) were subjected to RT-qPCR. After 50 µM of Teb was used to treat embryos at 12 hpf for 24 h, selected ZsGreen-positive embryos were cultured under the Teb-minus condition for the indicated period. Error bars stand for the standard error of the mean. f Measurement of luciferase activity in HEK293 cells after transiently transfecting the indicated combination of plasmids. The experimental conditions are identical to those in (c). Driver ‘10’ in (a) without EcR strongly stimulated the luciferase reporter activity irrespective of the presence of Teb, the difference of which was statistically non-significant. Luciferase activity was measured in triplicate or more. P-values were indicated in each figure.