Short hairpins based on miR-122 designed to express a particular siRNA guide strand. An siRNA sequence used previously to silence firefly luciferase (FLuc) was placed in the miR-122 stem-loop structure as shown schematically. a. Top: The sequence of the FLuc target mRNA (shown 3' to 5') was used to extend the complementary siRNA guide strand (middle underlined). Bottom: Schematic representation of the two short hairpins designed to express firefly luciferase siRNA. The 19 nt sequence of the FLuc siRNA guide strand is underlined. The sequence of the miRNA expected from these short hairpins is 23 nt in length (black). FLuc-ShM contains a mismatch (magenta). Sequence residues originating from the miR-122 pre-miRNA are blue. All hairpin forms are designed to produce identical guide strand sequences. b. Inducible silencing of firefly luciferase. FLuc short hairpin constructs were transiently transfected into NIH3T3-47 cells along with the Fluc reporter plasmid pGL3Luc and pCMV-lacZ as a transfection control. Cells were treated with 0.5 μM RSL1 or DMSO for 48 h, and cell lysates were subsequently assayed for firefly luciferase and β-galactosidase for normalization. The luciferase activity remaining 48 h after induction is shown as a percent of activity from matched transfected non-induced cells. Results represent the mean (+/- SD) of 3 experiments. p-values represent comparison of percent remaining activity for FLuc short hairpins vs. non-targeting control miR-122 short hairpins. * p < 0.01, ** p < 0.001.
Shea and Tzertzinis BMC Biotechnology 2010 10:76 doi:10.1186/1472-6750-10-76