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Fig. 2 | Journal of Animal Science and Biotechnology

Fig. 2

From: An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing

Fig. 2

Comparison of promoters controlling transactivator expression. a Schematic representation of five plasmids with different promoters, namely, PPGK, PCMV, PEF1α, PSV40, and PUbc, for controlling the expression of the transactivator. B~I: The inducible efficiency of five plasmids (p2, p13-p16). HEK293A (b), HeLa (d), A549 (f) and mIMCD3 (h) cells were transfected with each of the five plasmids with firefly luciferase as a reporter gene, respectively. Cells were grown in the absence or presence of Dox. Then, cells were harvested for luciferase activity assay. Firefly luciferase activity was normalized to Renilla luciferase activity. The fold induction of luciferase activity of these plasmids in HEK293A (c), HeLa (e), A549 (g) and mIMCD3 (i) cells were calculated as the ratio between the induced expression level with Dox and the background expression level without Dox. j-m The inducible efficiency of the five plasmids assessed by lentiviral infection. Cells of hPASMC (j) and A549 (l) were infected with corresponding lentivirus. The stable cell lines were induced with Dox. Then, equal numbers of stable cells were assayed for luciferase activity. Firefly luciferase values were normalized to the copy number of luciferase integrated into the genome of each stably cell line, which was determined by quantitative PCR. The fold induction of luciferase activity in hPASMC (k) and A549 (m) were also calculated. n The Dox sensitivity of the inducible system. HEK293A cells transfected with plasmids p1 and p2 were grown under different concentrations of Dox (0, 10, 50, 100, 500, 1,000, 2,000, and 4,000 ng/mL), and subjected to a luciferase activity assay 48 h after transfection. o The fold induction of luciferase activity between plasmids p1 and p2

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