Figure 4
Detection of two distinct miRNAs using triple-fluorescence-based SeVdp-miR-Sensor. (a) Structure of the SeVdp genome encoding E2-Crimson, KR, Hygr, and EGFP. Four copies of the complementary sequence for miR-302a, miR-17, or let-7a were incorporated into the 3′ UTRs of the KR and EGFP genes to construct the SeVdp-302aT/let-7aT and SeVdp-302aT/17T vectors. As a control, the SeVdp-FlucTx2 vector containing four copies of the complementary sequence for a portion of the firefly luciferase gene was used. (b) Evaluation of miRNA expression in NHDFs. NHDFs were infected with SeVdp-FlucTx2 (Control), SeVdp-302aT/let-7aT (302aT/let-7aT), or SeVdp-302aT/17T (302aT/17T), and the expression of EGFP, KR, and E2-Crimson (Crimson) was examined by fluorescence microscopy at 2 days post-infection. Images with pseudo-colour [green (EGFP), red (KR), and magenta (Crimson)] are indicated. Scale bars: 200 μm. (c) Evaluation of miRNA expression in hiPSCs. hiPSCs were infected with one of the vectors, and fluorescence was analysed as described for (b). (d) Quantitative comparison of miRNA expression in NHDFs. EGFP and KR expression in E2-Crimson( + ) NHDFs was analysed by flow cytometry at 4 days post-infection. EGFP and KR levels in cells infected with SeVdp-FlucTx2 were set to 1.0, and the relative MFI was evaluated. Data are presented as the mean ± standard deviation of three independent experiments. **P < 0.001 versus Control. (e) Quantitative comparison of miRNA expression in hiPSCs. Experimental procedures and values are the same as described for (d).
