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. 2015 Oct 6;10(11):1006–1013. doi: 10.1080/15592294.2015.1091145

Figure 1.

Figure 1.

Construction of a tetracycline-inducible TET-mediated global DNA demethylation system in HEK293T cells. (A) Schematic description of constructing a tetracycline-inducible TET-mediated global DNA demethylation system. The entire system was established through 2 main steps: Cloning TET1-CD into the lentiviral pTRIPZ vector which is equipped with a Tet-On system for transgene overexpression in the presence of Dox, and transfecting pTRIPZ-TET1-CD into HEK293T cells and developing single cell clones. (B) Dox treatment dose-dependent induced expression of TET1-CD and mTET1-CD in D1 and mC3 clone cells, respectively. The cells were harvested 24 h after Dox treatment. (C) DNA dot blot assay showed that Dox treatment dose-dependently induced genomic 5hmC production only in D1 cells. The cells were harvested 24 h after Dox treatment. (D and E) Dox treatment dose-dependently induced DNA demethylation in LINE-1 (D) and 4 selected genomic loci (E) in D1 clone cells but not mC3 clone and HEK293T parental cells. The cells were treated with Dox for 3 days. Bisulfite-pyrosequencing was used for DNA methylation analysis. Error bars represent SD from 3 independent experiments.