Volume 15, Issue 5, Pages 938-945 (May 2007) A Facile Lentiviral Vector System for Expression of Doxycycline-Inducible shRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha  Lars Aagaard, Mohammed Amarzguioui, Guihua Sun, Luis C Santos, Ali Ehsani, Hans Prydz, John J Rossi  Molecular Therapy  Volume 15, Issue 5, Pages 938-945 (May 2007) DOI: 10.1038/sj.mt.6300118 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Design and validation of the pFRT-based inducible knockdown system. (a) Insertion of a single tet repressor operon (tetO) into the 3′ end of the human U6 promoter using a pFRT plasmid backbone. Transcriptional start site indicated by uppercase G (bold), which is re-introduced by oligo-mediated short hairpin RNA (shRNA) cloning using the engineered BglII site (underlined). (b) Western blot analysis of protein kinase R (PKR) in HEK293 Flp-In TRex cells stably transfected with the pFRT plasmid utilizing either U6tetO- or H1tetO-promoted shRNAs targeting PKR, with or without doxycycline (dox) induction (3 days). (c) Northern blot analysis of Drosha mRNA in HEK293 Flp-In TRex cells stably transfected with the pFRT plasmid utilizing U6tetO-promoted shRNAs targeting Drosha, with or without dox induction. GAPDH was probed as an internal control. (d) Dose-dependent silencing of Drosha mRNA by northern blot analysis (upper panel) and northern analysis of induction of Drosha-targeting shRNAs (probing for the siRNA guide strand). Molecular Therapy 2007 15, 938-945DOI: (10.1038/sj.mt.6300118) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Design and validation of lentiviral-based inducible knockdown system in transduced HEK293 cells. (a) Northern analysis of siRNA guide strand expression with varying doxycycline (dox) doses and induction times. Lanes 1–2, un-induced; lanes 3–6, induced with 37, 111, 333, and 1,000 ng/ml dox for 48 hours, respectively; lanes 7–10, 1,000 ng/ml dox induction for 24, 12, 6, and 3 hours, respectively. U6 snRNA was probed as an internal RNA loading control. A long-term exposure of lanes 1–3 is shown in the lower panel. (b) Dox-dependent silencing of a transiently transfected shDr-sensitive Renilla luciferase reporter (psiCheck-Dr) after standardization to the internal Firefly luciferase control. Cells treated with 1 μg/ml dox 24 hours before reporter transfection. Values relative to un-treated shPKR transduced cells. (c) Schematic diagram of the pHIV-7-derived lentiviral vector pTIG (pHIV7-TetR-IRES-GFP) encoding a U6tetO-promoted short hairpin RNA (shRNA) cassette. CMV, cytomegalovirus promoter; eGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; psi, packaging signal; RRE, Rev response element; TetR, tetracycline regulatory protein; WPRE, woodchuck hepatitis virus post-transcription regulatory element. Molecular Therapy 2007 15, 938-945DOI: (10.1038/sj.mt.6300118) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Silencing of endogenous Drosha in pTIG-U6tetO-transduced HEK293 cells. (a) Real-time quantitative polymerase chain reaction (PCR) of Drosha messenger RNA (mRNA) after prolonged dox treatment (1 μg/ml for 8 days) after normalization to GAPDH mRNA. Values are relative to un-treated shPKR-transduced cells. (b) Western blot analysis of Drosha protein after prolonged dox treatment. Molecular Therapy 2007 15, 938-945DOI: (10.1038/sj.mt.6300118) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Reduction of mature microRNAs (miRNAs) after Drosha knockdown in pTIG-U6tetO-transduced HEK293 cells. (a) Northern blot analysis of mature miRNAs expressed after prolonged doxycycline (dox) treatment (1 μg/ml for 8 days). U6 snRNA was probed as an internal RNA loading control. (b) Dox-dependent de-repression of a transiently transfected miR-21-sensitive Renilla luciferase reporter that can either be cleaved by RISC (psiCheck-miR21-Perfect) or undergo miRNA-mediated translational inhibition in an miR-21-dependent manner (psiCheck-miR21-3xBulge). Cells were treated for 8 days with 1 μg/ml dox before reporter transfection. Renilla luciferase values were standardized to the internal Firefly luciferase control and represented relative to un-treated shPKR-transduced cells. P value indicated. (c) Phase contrast images (×100 magnification) of pTIG-U6tetO-shDr-transduced cells grown in the presence or absence of doxycycline for 8 days, then seeded at equal density (low = 5.3 × 105 cells per cm2, high = 1.6 × 106 cells per cm2) and subsequently grown for an additional 2 days. Hemocytometer counts of trypan blue–excluded cells 48 hour after seeding are indicated for the “low density” seeded cells (cells per cm2 ± SD, n = 3). Molecular Therapy 2007 15, 938-945DOI: (10.1038/sj.mt.6300118) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions