Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference

Abstract

RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a lentivirus vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As lentivirus vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.

FIG. 1.

(Left panels) A lentivirus vector-based system for conditional gene suppression with DOX-inducible siRNAs. (A) Schematic drawing of lentivirus vector plasmids used in this work. Cassettes consisting of the H1 promoter without (LV-H) or with (pLV-TH) the upstream tetO sequence, H1-siRNA (LV-Hsi), and tetO-H1-siRNA (LV-THsi) were cloned in the 3′ U3 region of pWPXL (http://www.tronolab.unige.ch/). All of the vectors contain an internal marker cDNA under transcriptional control of the EF-1α promoter. (B) Double-copy design of siRNA lentivirus vectors. During reverse transcription, the U3 region of the 5′ LTR is synthesized by using its 3′ homologue as a template, which results in a duplication of the siRNA cassette in the provirus integrated in the genome of transduced cells.

FIG.2.

(Right panels) Mode of action of the DOX-controllable transrepressor. (A) In the absence of DOX, tTR-KRAB binds to tetO and suppresses H1-mediated siRNA transcription, thus allowing normal expression of the cellular target gene (on). (B) In the presence of DOX, tTR-KRAB cannot bind to tetO and hence siRNAs are produced, leading to downregulation of their target (off). The internal marker contained in the siRNA vectors provides an inverse monitoring device, as it is on in the presence of DOX and off in its absence.

FIG. 3.

Regulation of GFP expression by using DOX-inducible siRNA. (A) HeLa cells carrying a single copy of a lentivirus vector expressing GFP from the EF-1α promoter (HeLa-GFP) were transduced with a control lentivirus vector (LV-TH) or with vectors producing a GFP-specific siRNA in a constitutive (LV-Hsi) or regulated (LV-THsi) manner, with or without LV-tTR-KRAB (lacking the internal ribosome entry site-dsRed cassette) and/or DOX as indicated. A truncated form of NGFR (ΔNGFR) served as an internal reporter in the siRNA vectors. (B) Conditional expression of the internal marker gene. HeLa-GFP cells dually transduced with LV-THsi/GFP and LV-tTR-KRAB were maintained in the presence or absence of DOX before FACS analysis with a monoclonal antibody specific for the extracellular domain of NGFR.

FIG. 4.

Regulation of endogenous genes by using DOX-inducible siRNAs. Left panels, downmodulation of p53. MCF-7 cells were infected with the indicated lentivirus vectors as described in Materials and Methods. Western blotting was performed with monoclonal antibodies against p53, GFP, or actin (as a control). Right panels, downmodulation of lamin A/C. The same experiment as for the left panels was performed with HeLa cells, using lamin-specific siRNA vectors and antibodies.

FIG.5.

Kinetics and dose responsiveness of DOX-inducible RNA interference. (A) MCF-7 cells were cotransduced with LV-THsi/p53 and LV-tTR-KRAB as described in Materials and Methods. Five days later, DOX was added at a concentration of 5 μg/ml. Cells were harvested just before DOX treatment (lane 0) and then at indicated time points. wt, nontransduced cells. Whole-cell extracts were analyzed by Western blotting with p53-specific antibodies. (B) At 5 days posttransduction as described for panel A, cells were placed in medium containing the following concentrations of DOX (in micrograms per milliliter): 0 (lane 1), 0.0005 (lane 2), 0.001 (lane 3), 0.002 (lane 4), 0.004 (lane 5), 0.008 (lane 6), 0.016 (lane 7), 0.063 (lane 8), 0.25 (lane 9), 1 (lane 10), and 5 (lane 11). wt, nontransduced cells. Western blot analyses of whole-cell extracts were performed after another 5 days.