Conditional Suppression of Cellular Genes: Lentivirus Vector-Mediated Drug-Inducible RNA Interference

RNA interference has emerged as a powerful technique to downregulatethe expression of specific genes in cells and in animals, thusopening new perspectives in fields ranging from developmentalgenetics to molecular therapeutics. Here, we describe a methodthat significantly expands the potential of RNA interferenceby permitting the conditional suppression of genes in mammaliancells. Within a lentivirus vector background, we subjected thepolymerase III promoter-dependent production of small interferingRNAs to doxycycline-controllable transcriptional repression.The resulting system can achieve the highly efficient and completelydrug-inducible knockdown of cellular genes. As lentivirus vectorscan stably transduce a wide variety of targets both in vitroand in vivo and can be used to generate transgenic animals,the present system should have broad applications.

INTRODUCTION

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Introduction

The externally controllable expression of exogenous cDNAs canbe readily obtained in cells or in animals owing to techniquespioneered more than a decade ago (2, 8). Recently, it was demonstratedthat the knockdown of endogenous genes could be achieved byRNA interference, and plasmid- or viral vector-based deliverysystems for the stable expression of small interfering RNAs(siRNAs) were rapidly created (1, 3, 5, 7, 9, 17). In many situations,however, it is desirable to suppress genes in a regulated fashion,for instance, to study cellular factors that play essentialroles during differentiation or development. On the basis ofthis premise, we created a lentivirus vector-based system fordrug-inducible production of siRNAs in stably transduced mammaliancells.

MATERIALS AND METHODS

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Materials and Methods

Vector construction. Vectors were constructed by using standard cloning procedures.The pSUPER and pSUPER-p53 constructs were described previously(5). pSUPER-siGFP was provided by F. Iseni (Geneva, Switzerland),and pSUPER-siLamin was a gift from R. Oggi (Lausanne, Switzerland).pLV-H was constructed by inserting the H1 promoter from pSUPERinto the 3' long terminal repeat (LTR) of pWPXL (http://www.tronolab.unige.ch/).To construct pLV-TH, the tetO cassette was excised from pUHD13-3(obtained from H. Bujard, Heidelberg, Germany) and cloned intopLV-H, upstream of the H1 promoter. Finally, the H1 promotercassette in pLV-H and pLV-TH was replaced by the H1-siRNA cassetteexcised from pSUPER-siRNA, generating pLV-H/siRNA and pLV-TH/siRNA,respectively. The sequence encoding tTR-KRAB (kindly providedby P. Lorenz and H.-J. Thiesen, Rostock, Germany) was clonedinto pWPXL, replacing the green fluorescent protein (GFP) marker(pLV-tTR-KRAB), or as part of a bicistronic unit also encodingDiscosoma sp. Red, using the encephalomyocarditis virus 5' internalribosome entry site.

The lentivirus vectors described here are available upon request(www.tronolab.unige.ch/).

Cell culture and transduction with lentivirus vectors. The 293T, HeLa, and MCF-7 cell lines were cultured in Dulbecco'smodified Eagle's medium supplemented with 10% fetal calf serum.All recombinant lentiviruses were produced by transient transfectionof 293T cells according to standard protocols (21). Briefly,subconfluent 293T cells were cotransfected with 20 µgof a plasmid vector, 15 µg of pCMV- ${Delta}$ R8.91, and 5 µgof pMD2G-VSVG by calcium phosphate precipitation. After 16 hmedium was changed, and recombinant lentivirus vectors wereharvested 24 h later.

To analyze the regulation of GFP, a HeLa cell clone carryinga single copy of the WPXL-GFP provirus (HeLa-GFP) was used.For transduction, HeLa-GFP, MCF-7, or HeLa cells were platedon 24-well plate (20 x 10⁴ cells/well), and after 16 h mediumcontaining recombinant lentivirus vectors was added. Following16 h of incubation, the cells were washed and split, and doxycycline(DOX) was added to half of the transduced cells at a final concentrationof 5 µg/ml. Five days later the cells were harvested andanalyzed by fluorescence-activated cell sorting (FACS).

Western blotting. Cell extracts were prepared in radioimmunoprecipitation assaylysis buffer (25 mM Tris [pH 7.5], 1% Triton X-100, 0.5% sodiumdeoxycholate, 5 mM EDTA, 150 mM NaCl) containing a cocktailof protease inhibitors (Sigma). The protein samples (10 µg)were separated on sodium dodecyl sulfate-4 to 20% gradient polyacrylamidegels, electroblotted to polyvinylidene difluoride membranes(Perkin-Elmer), and exposed to antibodies against p53 (SantaCruz Biotechnology), lamin A/C (Santa Cruz Biotechnology), GFP(Clontech), and actin (Calbiochem). Antibodies conjugated withhorseradish peroxidase (Amersham) and enhanced chemiluminescence(Amersham) were used for detection.

FACS analysis. Harvested HeLa-GFP cells transduced with lentivirus vectorscarrying ${Delta}$ NGFR cDNA were incubated with monoclonal antibody specificfor human nerve growth factor receptor NGFR (Becton DickinsonPharMingen) labeled with phycoerythrin, washed twice, and analyzedwith a FACSscan (Becton Dickinson) for green (GFP) and red (NGFR-phycoerythrin)fluorescence. MCF-7 and HeLa cells cotransduced with LV-THsi/p53or LV-THsi/lamin and pLV-tTR-KRAB-Red and cultured in presenceor absence of DOX were harvested and analyzed with a FACSscanfor green and red (dsRed) fluorescence.

Immunofluorescence. MCF-7 and HeLa cells cotransduced with LV-THsi/p53 or LV-THsi/laminand pLV-tTR-KRAB-Red and cultured for 5 days in the presenceor absence of DOX were fixed with methanol (10 min, -20°C),blocked with phosphate-buffered saline-1% bovine serum albumin,and stained with antibodies against p53 (Santa Cruz Biotechnology)or lamin A/C (Santa Cruz Biotechnology), using secondary antibodiesconjugated with Alexa 633 (Molecular Probes) for detection.Images were acquired by using three-color confocal microscopy(LSM 510; Carl Zeiss) and analyzed with Zeiss software.

RESULTS AND DISCUSSION

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Results and Discussion

We took advantage of a tetracycline-controlled hybrid protein,tTR-KRAB, in which the tetracycline repressor (tTR) from Escherichiacoli Tn10 is fused to the KRAB domain of human Kox1 (6, 8).KRAB is an approximately 75-amino-acid transcriptional repressionmodule found in many zinc finger-containing proteins, whichcan suppress, in an orientation-independent manner, both polymeraseII- and polymerase III-mediated transcription within a distanceof up to 3 kb from its binding site, presumably by triggeringthe formation of heterochromatin (4, 6, 12, 14, 19). When linkedto the DNA-binding domain of tTR, KRAB can modulate transcriptionfrom an integrated promoter juxtaposed with tet operator (tetO)sequences (6). In the absence of DOX, tTR-KRAB binds specificallyto tetO and suppresses the activity of the nearby promoter.Conversely, in the presence of DOX, tTR-KRAB is sequesteredaway from tetO, thus permitting gene expression (6).

We used human immunodeficiency virus type 1-derived lentivirusvectors (designated LV) as delivery vehicles because we aimedfor a system that would be easy to apply to a broad varietyof cellular targets, both ex vivo (cell lines, primary cellsincluding stem cells, fertilized oocytes, and blastocysts) andin vivo (e.g., brain and liver) (9, 13, 15, 16-18, 20), andbecause tetO-linked transcriptional units are repressed by tTR-KRABonly when integrated in the genome. The tTR-KRAB cDNA was expressedfrom the ubiquitously active EF1- ${alpha}$ promoter as part of a bicistronictranscript also producing the dsRed marker (Fig. 1A, LV-tTR-KRAB).The regulated siRNA vectors were constructed by inserting atetO-H1 promoter-siRNA cassette into the U3 region of the 3'LTR of a self-inactivating lentivirus vector (Fig. 1A, LV-THsi).During reverse transcription, the vector RNA 3' U3 region servesas the template for the synthesis of its 5' DNA homologue, sothat the tetO-H1-siRNA cassette is duplicated in the integratedprovirus (Fig. 1B). We chose this double-copy configurationto obtain higher rates of siRNA synthesis. Sequences encodingsiRNA hairpin precursors were designed as described previously(5). Control vectors carried either a constitutively activeH1-siRNA cassette (LV-Hsi) or the H1- or tetO-H1 transcriptionalelements without downstream siRNA-coding sequence (LV-H andLV-TH, respectively). All siRNA and control vectors also carrieda marker gene downstream of an internal EF1- ${alpha}$ promoter. We predicted(Fig. 2A) that cells cotransduced with LV-THsi and LV-tTR-KRABwould normally express the gene targeted by the siRNA when maintainedin the absence of DOX, owing to tTR-KRAB-mediated suppressionof siRNA synthesis. In contrast, addition of the drug wouldrelieve this inhibition and allow for target gene downregulation(Fig. 2B). Expression of the internal marker gene would alsobe subjected to conditional tTR-KRAB repression, thus providingan internal monitoring device.

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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-1 ${alpha}$ 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.

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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.

In a first series of experiments, we probed the ability of thissystem to regulate the production of GFP in HeLa cells stablyexpressing this fluorophore (Fig. 3A). Vectors were used ata multiplicity of infection of 10 to ensure good rates of (co)transduction.HeLa-GFP cells transduced with the empty LV-TH vector remainedstrongly GFP positive irrespective of their culture conditions.In contrast, cells transduced with the constitutively activeLV-Hsi/GFP vector exhibited a strong downregulation of the marker.In cells transduced with the controllable LV-THsi/GFP vector,GFP expression was observed only in the presence of tTR-KRABand in the absence of DOX (Fig. 3A). Correspondingly, in theabsence of drug, tTR-KRAB suppressed the expression of the vector's ${Delta}$ NGFR internal reporter gene (Fig. 3B). As expected, the tTR-KRAB-mediatedsuppression of siRNA production was equally efficient whethertetO was inserted in the sense or antisense orientation andupstream or downstream of the H1 promoter (data not shown).

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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-1 ${alpha}$ 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 ( ${Delta}$ NGFR) 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.

Next, we tested our system for the regulation of truly endogenousgenes. We the chose p53 and lamin genes as targets because highlyeffective siRNAs directed against these genes were previouslyidentified and well characterized (5, 7). MCF-7 breast cancercells were used as substrates for p53 downregulation studies(Fig. 4, left panels). Cells cotransduced with LV-tTR-KRAB andLV-THsi/p53 produced wild-type levels of p53 when cultured inthe absence of DOX, indicating full repression of siRNA synthesis(lower blot, lane 7). This repression was mediated by tTR-KRAB,since p53 was undetectable in cells transduced only with LV-THsi/p53,whether or not DOX was present in the culture medium (upperblot, lanes 7 and 8). In contrast, addition of the drug to thedually transduced cells resulted in rates of p53 downmodulationas robust as those observed in cells containing the constitutivelyactive LV-Hsi/p53 vector (compare lane 8 in the lower blot withlanes 5 and 6 in both blots). Similar results were obtainedfor lamin in HeLa cells transduced with the corresponding siRNAlentivirus vectors (Fig. 4, right panels). It is noteworthythat in both settings the drug-induced production of the siRNAs,and hence the suppression of the p53 or lamin target gene, correlatedwith the expression of the lentivirus vector internal GFP marker,whether examined by Western blotting (Fig. 4) or by FACS orconfocal microscopy (data not shown).

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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.

Taken together, these results indicate that the tTR-KRAB-regulated,lentivirus vector-mediated delivery of siRNAs allows for thecontrollable suppression of cellular genes both with a highdegree of efficacy and without significant leakiness. To completethe characterization of this system, we defined its kineticsand DOX dose responsiveness (Fig. 5). We chose p53 as targetfor these analyses because the half-life of this protein isrelatively short, around 12 h. In MCF-7 cells dually transducedwith the LV-THsi/p53 and LV-tTR-KRAB vectors, p53 steady-statelevels started to decrease as early as 12 h after addition of5 µg of DOX per ml to the culture medium and became undetectableby Western blotting within 36 h (Fig. 5A). This suggests thatRNA interference was fully effective in less than 24 h, implyingthat the DOX-mediated sequestration of tTR-KRAB rapidly unleasheshigh rates of siRNA production from the integrated H1 promoters.A dose-response analysis further revealed an extreme sensitivityto DOX control, while pointing to the possibility of some tuningof the gene suppression. Indeed, whereas p53 downregulationwas already apparent at the low DOX concentration of 0.004 µg/ml,full-blown suppression was achieved only at a dose of 0.25 µg/ml(Fig. 5B). The anti-p53 siRNA used in this experiment beingvery efficient, a greater range of DOX concentrations may allowfor a modulation of the degree of gene knockdown with siRNAsof lower specific activity.

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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.

In summary, we provide a system for the conditional suppressionof genes in mammalian cells. The versatility of its mode ofdelivery suggests very broad uses, as lentivirus vectors cantransduce a wide range of targets, including stem cells, andcan be used for generating transgenic animals from several species.In the latter setting, the system described here should offersignificant advantages over currently available conditionalknockout techniques, among which are its reversibility and simplicityof use. While the lentivirus vector-mediated delivery of drug-inducibleRNA interference may thus be of particular interest for thestudy genes involved in development and differentiation, itis likely to be useful in many other areas of biology as well.

ACKNOWLEDGMENTS

We thank P. Lorenz and H.-J. Thiesen for tTR-KRAB cDNA, F. Isenifor pSUPER-siGFP, R. Oggi for pSUPER-siLamin, S. Vianin fortechnical assistance, and M.-O. Sauvain, other members of ourlaboratory, and J. Szulc for helpful discussions.

This work was supported by the Swiss National Science Foundationunder the auspices of the National Center for Competence inResearch Frontiers in Genetics program and by the Institut Claytonde la Recherche.

FOOTNOTES

* Corresponding author. Mailing address: Department of Genetics and Microbiology, CMU, Faculty of Medicine, University of Geneva, 1 rue Michel-Servet, 1211, Geneva 4, Switzerland. Phone: 41 22 702 5720. Fax: 41 22 702 5721 or 41 22 702 5702. E-mail: didier.trono{at}medecine.unige.ch.

REFERENCES

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