Figure 4

Generation and characterization of the ability of I¹⁵⁵, a mammalian expression vector encoding sequences partially antisense to miR-155, to restore GFP expression in a DC line cotransfected with bic¹⁵⁵and GFP/miR-155as. A, Schematic diagram to depict I¹⁵⁵, a DNA plasmid construct that contains 6 tandemly repeated sequences partially antisense to miR-155 cloned into the 3' UTR of red fluorescent protein. The partially complementary sequences of I¹⁵⁵shown in the diagram bind imperfectly to miR-155 to form a 4-nucleotide mismatched bulged site and may serve as a competitive inhibitor of miR-155. B, Fluorescence microscope image at 24 hrs of DC-1 cells transfected with GFP/miR-155as in combination with a control construct encoding non-specific miRNA (panel I), bic¹⁵⁵(panel II) or both bic¹⁵⁵and I¹⁵⁵(panels III and IV). The empty pcDNA3 vector was used to standardize the total amount of transfected DNA. GFP/miR-155as and bic¹⁵⁵transfection complexes were formulated in Lipofectamine 2000 in a 1:9 mass ratio. The transfection efficiency of I¹⁵⁵was measured by the red fluorescent intensity (panel V) and the degree to which I¹⁵⁵restored GFP expression in bic¹⁵⁵- and GFP/miR-155as-cotransfected cells (panel IV). To further characterize the I¹⁵⁵construct, DC-1 cells were transfected with 0.1 μg of either RFP/control (panel V) or I¹⁵⁵(panel VI).