Figure 4

Retroviral delivery of the pHUSH system allows the inducible expression of shRNA, miRNA or proteins. (A) Vector diagram of the pHUSH retroviral system. Inducible pol II or pol III expression cassettes are introduced by Gateway^® recombination. (B) Improved p53 knockdown by CMV-miRNA in comparison to H1-shRNA vectors at low MOI. H1-shRNA and CMV-miRNA vectors with hairpins designed against murine p53 as described in additional file 2 were cloned into the pHUSH retroviral backbone. 3T3 cells were infected at an MOI = 0.2, selected in 1.5 ug/mL puromycin, and cultured ± 1 ug/mL Dox, Knockdown of p53 was determined by qRT-PCR at Day 4 post-Dox and was normalized to p53 levels in the absence of Dox. (C) Inducible expression of proteins by the pHUSH retroviral system. Inducible EGFP and EGFP-MELK-D150A expression cassettes were cloned into the pHUSH retroviral backbone. HCT116 cells were infected with the appropriate viral construct, selected in 5 ug/mL puromycin, and treated ± 1 ug/mL Dox for 72 h. Equal total protein was fractionated by non-reducing SDS-PAGE and imaged by the Typhoon fluorescent imager at the indicated excitation and emission spectra (WB = western blot). Equal loading was confirmed by western blotting with an anti-tubulin antibody.