Dual gene silencing activity of linear tsiRNAs, tsiER(s25a21), tsiER(s21a21), and tsiER(s15a21), against the EGFP and RLuc genes. (A) The predicted structure of tsiER RNAs. (Boxes) Antisense strands of (green) siE and (red) siR genes. In these pGD vector constructs, the green antisense strands of siE duplexes are expressed from the U6 promoter, whereas those of (red) siR are from the H1 promoter (see also Fig. 1A). Probe sequences for detection of siE and siR antisense strands are underlined. (B) Luciferase assay following transfection of three cellularly expressed tsiRNA constructs, in which the siE and siR components are combined in opposing transcriptional directions. FLuc and RLuc expression levels of cells transfected with the plasmid encoding control tsiRNA (tsiC) were set at 100%. Relative means (+SD) from three independent experiments are shown. (C) Northern blot analysis for detection of antisense strands of siE and siR siRNAs. Detection of cellular snU6 RNA was used as a loading control. (Arrows) Processed 21–23-nt antisense strand (AS) siRNA products (siE and siR). Probe sequences and positions are described in A. (Right) Marker sizes (in nucleotides, nt).
