Figure 3.

Generation of four effective individual siRNAs from a single dlhRNA transcript containing two adjacent lhRNAs. (A) Schematic representation of a dlhRNA expression cassette driven by a single promoter showing the predicted structure and derivation of four siRNAs. (B) Effective dual targeting long hairpin RNAs lhtat-nef +1 and lhLTR-int +1 were both combined in 5′ or 3′ positions within the dlhRNA transcript to generate lhLI-TN and lhTN-LI. (C) Low molecular weight northern blot analysis was carried out on total RNA extracted from cells transfected with the dlhRNA expression cassettes with individual targeting shRNAs used as positive controls. Labelled probes complementary to the guide and antisense strand of LTR, int, tat and nef were hybridized to immobilized RNA and exposed to a phosphorimaging plate. Precursor hairpin RNAs as well as processed siRNAs are indicated. The amount of processed guide strand is shown and normalized for each blot relative to the shRNA (set at 100). Decade Marker^TM indicates fragment size and a probe complementary to small nuclear U6 RNA was used to detect U6 snRNA as a loading control. (D) Dual luciferase reporter assays showing knockdown of the sense (S) and antisense (AS) targets of LTR, int, tat and nef when the target sequence was inserted downstream of the Renilla luciferase open reading frame. Values represented are mean ratios of Renilla to Firefly luciferase (n = 3, ± SEM) and are normalized to cells transfected with a plasmid containing a U6 promoter only with no RNAi effector sequence (mock).