Figure 1

Strategy for generating intracellular siRNAs and effect of the expressed siRNAs on transgene expression. (A) The convergent U6 expression cassette encodes sense (black) and antisense RNAs (red) that terminate at directional termination sequences (represented as five consecutive thymidines [T]). The complementary RNAs anneal and undergo further Dicer-dependent processing to produce functional siRNAs. A U6 convergent expression vector containing an EGFP-specific insert (DualU6GFP) reduces dEGFP-mediated cell fluorescence as measured by (B) flow cytometry and (C) fluorescence microscopy. This same expression vector suppresses both (D) dEGFP protein levels and (E) dEGFP RNA levels. For Western analysis, β-actin protein levels were used as a loading control. For RNA analysis, the control for loading was 18s rRNA.