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Fig. 3 | Journal of Animal Science and Biotechnology

Fig. 3

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

Fig. 3

Precise regulation of miRNA expression and its target. a Schematic representation of p2-based inducible miRNA expression plasmid. The EGFP and the downstream primary miRNA sequence were positioned under the control of the TREs. b Inducible miRNA expression. HEK293A cells were infected with lentiviruses carrying the primary sequences of hsa-miR-210, hsa-miR-21, and hsa-miR-26a, and cultured in the absence or presence of 1 μg/mL Dox for 48 h. Cells were harvested and subjected to measurement of the miRNA level by qRT-PCR. Lentivirus without any primary miRNA sequence was used as a NC. The level of each miRNA was calculated as the fold change relative to that in NC without Dox. c The dose-dependent effect of miRNA inducibility. miR-210-3p and miR-21-5p were induced under different concentrations of Dox (0, 10, 50, 100, 500, 1,000, 2,000, and 4,000 ng/mL) for 48 h and subjected to measurement of the miRNA level. The level of each miRNA was calculated as the fold change relative to that without Dox. d The time-dependent and reversible effect of miRNA inducibility. miR-210-3p and miR-21-5p were induced with 1 μg/mL Dox for 0, 12, 24, 36, and 48 h. Subsequently, cells that had undergone 48-h induction were subcultured in fresh medium without Dox for another 24, 48, 72, and 96 h, and harvested for miRNA detection. e The PDCD4 protein levels of cells inducibly expressing miR-21-5p under different Dox concentrations were assayed by western blotting. Beta-actin served as a loading control. f The PDCD4 protein levels were quantified. The data are expressed relative to that without Dox, and are presented as mean ± SD (n = 3). ***P < 0.001

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