Figure 6

Constructed vectors for this study and in vitro target validation of gga-miR-200a-3p. A Schematic diagrams of expression vector for DsRed with gga-miR-200a-3p, eGFP with partial 3′-UTR of TGFβ2 and Luciferase with partial 3′-UTR of selected genes. The miR200a/DsRed vector expresses both red fluorescence protein (RFP) and gga-miR-200a-3p; TGFβ2/EGFP vector expresses both enhanced green fluorescence protein (eGFP) and cloned 3′-UTR of TGFβ2. The “Gene”/Luc vectors express both luciferase and cloned 3′-UTR of TGFβ2, MAP2K4, or ZAK and named as TGFβ2/Luc, MAP2K4/Luc, and ZAK/Luc, respectively. The two restriction sites for cloning are indicated at both sides of inserted gene. B miR-200a/DsRed and TGFβ2/eGFP were co-transfected into HD11 chicken macrophage cell line in a dual fluorescence reporter. After 48 h of co-transfection, eGFP and DsRed expression were examined under a fluorescence microscope. White bars indicate 400 µm in length. C Luciferase reporter assay conducted for 24 h following co-transfection of “Gene”/Luc vectors with miR200a/DsRed or luciferase vector with miR200a/DsRed as the control in the HD11 chicken macrophage cell line. Results (mean ± SEM) are representative of three independent experiments.