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. 2016 Sep 27;5(9):e367. doi: 10.1038/mtna.2016.74

Figure 2.

Figure 2

An EcR-based singular gene switch induces robust transgene expression with low background expression. (a) A schematic diagram of the pEUI(+) vector optimized for transgene expression under the control of a chemical inducer, tebufenozide. A single-nucleotide substitution of A to G at nucleotide position 4,783 was introduced to erase the EcoR I recognition sequence (arrow) without altering the amino acid sequence. The box shows the nucleotide sequences of the MCS of pEUI(+) with the restriction endonuclease recognition sites. (b) A schematic drawing of the singular pTet vector that responds to Tet. (c) Comparison of pEUI(+) and pTet vector capacities through the measurement of firefly Luc activity. Cells transfected with pEUI(+)-Luc show very low background expression (~1.7-fold increase versus pGL3-Basic) compared with pTet-Luc-transfected cells (~18.7-fold). pEUI(+)-Luc shows tebufenozide-dose-dependent transgene stimulation. Luc activity was normalized to cotransfected Renilla Luc activity. (d) The fluorescence intensity in pEUI(+)-EGFP-transfected cells increases in a tebufenozide-dose-dependent manner (e'–h'). The mini-Tol2-EGFP vector lacking a promoter was used for control transfection (a'–d'). HEK293T cells transfected with the indicated plasmid constructs were treated with tebufenozide (0–5 μmol/l) for 12 hours and then examined under a fluorescence microscope. Scale bar in a', 200 μm. EcR, ecdysone receptor; MCS, multiple cloning site; Tet, tetracycline.