The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

Abstract

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.

Fig. 1.

pINDUCER lentivirus elicits inducible cDNA and shRNA expression. (A) Diagrams of the pINDUCER vector series. R, tRFP; U, Ubc promoter; E, EF1-α promoter; L, luciferase; G, eGFP; P, puromycin resistance; N, neomycin resistance. (B) pINDUCER10 elicits robust cDNA expression and RNAi across polyclonal populations. U2OS osteosarcoma cells were transduced [multiplicity of infection (MOI) = 0.5] with pINDUCER10-shRB, selected for puromycin resistance, and cultured with or without dox for 4 d. Cells were analyzed for the tRFP fluorescence by flow cytometry (Left) or expression of Rb or Gapdh (loading control) proteins by Western blot (Right). (C) pINDUCER10 elicits effective RNAi in nontransformed cells. Multiple shRNAs targeting Rest were subcloned into pINDUCER10. Polyclonal populations of HMECs transduced with the indicated lentiviral shRNA were cultured with or without dox for the indicated times, and Rest or Vinculin (loading control) levels were determined by Western blot. Control indicates a nontargeting shRNA. (D) Kinetics of cDNA induction with pINDUCER20 (ORF-UN). Polyclonal populations of SW1417 colon cancer cells transduced with pINDUCER20-REST were cultured with or without dox for the indicated times, and Rest or Vinculin (loading control) protein levels were determined by Western blot. (E) Dose-dependent cDNA induction with pINDUCER20. Cells from D were cultured with the indicated dox concentration for 48 h, and Rest or Vinculin (control) levels were determined by Western blot.

Fig. 2.

Dual-fluorescent pINDUCER11 (miR-RUG) system improves inducible cDNA expression and RNAi across polyclonal populations. (A) Diagram of pINDUCER11 (miR-RUG). pINDUCER11 encodes a constitutive cassette (rtTA3 and eGFP) and an inducible transcript (shRNA and tRFP). (B) Isolation of cells with differing rtTA3/eGFP expression. Polyclonal populations of HMECs were infected with pINDUCER11 (MOI = 0.7) and FACs-sorted for indicated levels of eGFP fluorescence. (C) Stable eGFP fluorescence in pINDUCER11 transduced cells. Transduced HMECs from B were measured for mean eGFP fluorescence intensity by flow cytometry 14 d after the original FACs sorting. (D) Higher rtTA3/eGFP expression increases inducibility in polyclonal cell populations. HMECs from B were cultured in Dox for 4 d and measured for tRFP fluorescence by flow cytometry. The graph shows the fold induction of tRFP fluorescence (mean +dox tRFP fluorescence intensity/mean −dox tRFP fluorescence intensity). (E) Higher rtTA3/eGFP expression increases inducible RNAi in polyclonal cell populations. HMECs transduced with pINDUCER11-shREST and FACs-sorted for various levels of eGFP fluorescence were cultured with or without dox for 3 d and analyzed by Western blot for Rest or Vinculin (control) protein levels.

Fig. 3.

pINDUCER11 (miR-RUG) elicits robust inducibility in the mouse mammary gland and mammary tumors in vivo. (A) Mouse mammary glands were excised, dispersed into single-cell suspension, transduced with pINDUCER11 (miR-RUG), and retransplanted orthotopically into the cleared fat pad of syngeneic mice with or without dox. After 4 wk, mammary glands were explanted and visualized by fluorescence microscopy as indicated. (B) p53^−/− mouse mammary tumors were excised, dispersed into single-cell suspension, transduced with pINDUCER11, sorted for eGFP fluorescence, and retransplanted orthotopically into syngeneic mice. Upon tumor formation (>300 mm³), mice were treated with or without dox for 7 d, and tumors were explanted and visualized by phase or fluorescence microscopy as indicated. (C) MDA-MB-231 human breast cancer cells were transduced with pINDUCER11 and transplanted into the mouse mammary gland. Mice were treated with or without dox for 28 d. Tumors were dispersed into single-cell suspension and analyzed for fluorescence (eGFP and tRFP) by flow cytometry.

Fig. 4.

pINDUCER11 (miR-RUG) enables validation of tumor dependencies in vivo. (A and B) SUM159 human breast cancer cells were transduced with pINDUCER11-shBIRC4 at MOI = 3 and analyzed for (A) cellular fluorescence or (B) BIRC4 mRNA levels. (C) Cells from A were transplanted orthotopically into the mouse mammary gland. Mice were administered dox (+dox) or vehicle (−dox) and assessed for tumor volume over time. (D) Mice from C were observed for overall survival for the indicated times.