160–169www.elsevier.com/locate/gene

Gene 395 (2007)

Selection and validation of optimal siRNA target sitesfor RNAi-mediated gene silencing

Qing Luo a,b, Quan Kang a,b, Wen-Xin Song b, Hue H. Luu b, Xiaoji Luo a,b, Naili An c,Jinyong Luo a,b, Zhong-Liang Deng a,b, Wei Jiang b, Hong Yin b, Jin Chen a,b, Katie A. Sharff b,

Ni Tang a,b, Erwin Bennett b, Rex C. Haydon b, Tong-Chuan He a,b,⁎

a The Children’s Hospital, and the Key Laboratory of Diagnostic Medicine designated by the Ministry of Education,Chongqing University of Medical Sciences, Chongqing 400016, China

b Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, 5841 South Maryland Avenue,MC3079, Chicago, IL 60637, USA

c The Scripps Research Institute, La Jolla, CA 92037, USA

Received 23 November 2006; received in revised form 13 February 2007; accepted 15 February 2007

Available onlin

Received by A. Rynditch

e 23 March 2007

Abstract

RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discoveries. Onemajor challenge of using RNAi is to identify the most effective short interfering RNAs (siRNAs) sites of a given gene. Although several publishedbioinformatic prediction models have proven useful, the process to select and validate optimal siRNA sites for a given gene remains empirical andlaborious. Here, we developed a fluorescence-based selection system using a retroviral vector backbone, namely pSOS, which was based on the premisethat candidate siRNAs would knockdown the chimeric transcript between GFP and target gene. The expression of siRNAwas driven by the opposingconvergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that GFPsignal reduction was closely correlated with siRNAknockdown efficiency of humanβ-catenin, as well as with the inhibition ofβ-catenin/Tcf4 signalingactivity. The pSOS should not only facilitate the selection and validation of candidate siRNA sites, but also provide efficient delivery tools of siRNAs viaviral vectors in mammalian cells. Thus, the pSOS system represents an efficient and user-friendly strategy to select and validate siRNA target sites.© 2007 Elsevier B.V. All rights reserved.

Keywords: Beta-catenin signaling; Expression knockdown; RNA interference; Short interfering RNA; Wnt signaling

1. Introduction

Double-stranded RNA (dsRNA) can induce gene-silencingprocesses in eukaryotes through the degradation of homologousmRNAs, a process known as RNA interference (RNAi) inanimals and post-transcriptional gene silencing (PTGS) inplants (Sharp, 1999; Bosher and Labouesse, 2000; Zamore,2001; Hannon, 2002; Paddison and Hannon, 2002; Tijsterman

Abbreviations: BC, β-catenin; GFP, green fluorescent protein; H1, RNApolymerase III (Pol III)-dependent promoter of the human H1 transcript of thenuclear RNase P complex; RFP, (monomeric) red fluorescent protein; RNAi,RNA interference; siRNA, short interfering RNA; U6, RNA polymerase III (PolIII)-dependent promoter of the human U1 small nuclear RNA (snRNA) gene.⁎ Corresponding author. Tel.: +1 773 702 7169; fax: +1 773 834 4598.E-mail address: tche@surgery.bsd.uchicago.edu (T.-C. He).

0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.gene.2007.02.030

et al., 2002; Dykxhoorn et al., 2003; Mello and Conte, 2004;Montgomery, 2004; Tian et al., 2004). Small synthetic 19–27nucleotide dsRNAs can act as short interfering RNAs (siRNAs)to mediate gene-specific silencing in mammalian cells (Sharp,1999; Bosher and Labouesse, 2000; Zamore, 2001; Hannon,2002; Paddison and Hannon, 2002; Tijsterman et al., 2002;Dykxhoorn et al., 2003; Scherer and Rossi, 2003; Mello andConte, 2004). Practical applications of RNAi have beenfacilitated by using expression vector-based strategies tointroduce siRNAs, commonly referred to as short hairpinRNAs (shRNAs), into mammalian cells using Pol III-dependentpromoters (Paul et al., 2002; Scherer and Rossi, 2003; Medema,2004; Aravin and Tuschl, 2005; Arziman et al., 2005; Bentwich,2005; Dillon et al., 2005; Huppi et al., 2005; Sandy et al., 2005;Echeverri and Perrimon, 2006). To date, two RNA polymerase

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III (Pol III) promoters, the human U6 small nuclear promoterand the human H1 promoter, are the most commonly used onesto drive the expression of shRNAs (Brummelkamp et al., 2002a,b; Lee et al., 2002; Paddison et al., 2002; Paul et al., 2002; Sui etal., 2002; Scherer and Rossi, 2003; Sui and Shi, 2005). The H1promoter is an RNA polymerase III (Pol III)-dependentpromoter of the H1 transcript of the nuclear RNase P complex,while the U6 is a Pol III-dependent promoter of the U1 smallnuclear RNA (snRNA) gene (Paul et al., 2002; Scherer andRossi, 2003). Although RNAi has great potential for reversegenetics and drug discoveries, a broader use of this techniquehas to overcome several practical challenges, such as selectionof effective target sites, efficient transfer of siRNAs into cells ortissues, and achieving controllable long-term silencing of targetgenes. Currently, there is no reliable and efficient way to predictoptimal siRNA sequences. As a result, multiple target sites of agiven gene have to be tested, and validation of silencingefficacy is laborious and time-consuming.

Here, we developed a simplified and efficient fluorescence-based screening and validation system, namely pSOS, to assessgene-silencing efficacy of siRNAs. The pSOS system has twoessential components, one of which expresses a chimerictranscript between GFP and the coding region of a target genedriven by a Pol II promoter, and the other expresses a siRNAduplex under the control of dual convergent Pol III promoters.Thus, upon introducing pSOS-based siRNA plasmids intomammalian cells, the reduction in GFP signal would reflect thesilencing efficiency of siRNAs. We demonstrated that theexpression of GFP was effectively silenced by using GFP-specific siRNA. As a proof-of-principle study, we tested seventarget sites of human β-catenin. Our results showed that thereduction of GFP signals was closely correlated with the siRNAknockdown efficiency of β-catenin expression and its signalingactivity. Although the dual Pol III promoter strategy or the useof reporter genes to assess silencing efficacy has been reported,the pSOS system is simplistic, versatile, and user-friendly.Furthermore, this system can be readily converted into viralvectors to deliver optimal siRNAs in vitro and in vivo.

2. Materials and methods

2.1. Cell culture and chemicals

HEK-293 and HCT116 cell lines were purchased fromATCC. HEK-293 cells were maintained in complete DMEMcontaining 10% FBS and 100 units/ml of penicillin, and 100 μg/ml of streptomycin. HCT116 cells were maintained in completeMcCoy's 5A medium containing 10% FBS and 100 units/ml ofpenicillin, and 100 μg/ml of streptomycin. Unless otherwiseindicated, all chemicals were purchased from Sigma or FisherScientific.

2.2. Construction of a dual-promoter vector for selectingoptimal siRNAs (pSOS)

Using PCR amplifications, we assembled a cassettecontaining human elongation factor 1α (hEF1α) promoter,

GFP coding region (with a stop codon), and a linker withmultiple cloning sites flanked with Pac I sites in MSCVretroviral vector backbone. The fragment containing the dualconvergent H1 and U6 Pol III promoters was constructed byPCR amplification. For efficient cloning and screening ofsiRNA cassettes, a three-enzyme linker flanked with Sfi I siteswas engineered between the convergent opposing promoters.The resultant siRNA expression vector was designated aspSOS (Fig. 1A). All PCR amplified sequences were verified byDNA sequencing. Full-length vector sequence of pSOS isshown in Supplementary Fig. 3 and available at: www.boneandcancer.org/resources.htm.

2.3. Construction of pSOS-based siRNA expression vectors

To construct the pSOS vector expressing GFP siRNA, weannealed three pairs of oligonucleotides, and subclonedinto the Sfi I site of pSOS, resulting in pSOS-siGFPs. As acontrol vector (or pSOS-siGFP1m), we introduced fourmutant nucleotides (underlined) in the siGFP1 target site,and constructed pSOS-siGFP1m. To construct pSOS vectorexpressing human β-catenin siRNA, we first subcloned thecoding region (267nt–2266nt, excluding the coding sequencefor the N-terminus destruction domain) of human β-catenininto Sal I and Bgl II sites of pSOS, resulting the pSOS-BC.We chose seven target sites within the coding region ofhuman β-catenin and subcloned the oligonucleotide cassettesinto the Sfi I site of pSOS-BC, resulting in pSOS-siBCvectors. It should be pointed out that two orientations (withrespect to H1 or U6 promoter-driven expression of the sense-strand) were obtained for each of the above target sites.Authenticity of PCR amplified sequences and the oligonucle-otide cassettes was verified by DNA sequencing. Cloning andconstruction details of the above vectors are available uponrequest.

2.4. Qualitative and quantitative measurement of fluorescence

HEK-293 cells were transfected with pSOS, pSOS-siGFP,pSOS-BC, or pSOS-siBC plasmids, along with pCMVβ, usingLipofectAMINE (Invitrogen) according to the manufacturer'sinstructions. At the indicated time points post transfection, theexpression of GFP was examined and recorded using afluorescence microscope. For quantitative measurement ofGFP fluorescence, the transfected cells were lyzed andsubjected to fluorescence measurement using the ModulusFluorometer (Turner Biosystems). Each assay condition wascarried out in triplicate. Fluorescence reading for each assay wasfurther normalized with the transfection efficiency (β-galacto-sidase activity).

2.5. β-Catenin/Tcf4 luciferase reporter assay

The reporter assay was conducted as previously described(He et al., 1998a, 1999; Zhou et al., 2002, 2003; Si et al., 2006).HEK-293 and HCT116 cells were seeded in 12-well plates andtransfected with 0.2 mg of pTOP-Luc, 0.1 μg of pCMVβ,

Fig. 1. A fluorescence-based vector system for selecting optimal siRNAs (pSOS). (A) Schematic representation of the pSOS vector. Expression of siRNA duplex isdriven by the convergent opposing Pol III promoters H1 and U6. Expression of GFP is driven by human EF1α promoter (hEF1). A polylinker site immediatelyfollowing the GFP coding region (with a stop codon) is designed to clone the gene of interest (GOI). (B) Detailed steps for subcloning siRNA oligonucleotide cassettesinto pSOS vector. Two strategies can be employed to construct a siRNA expression vector. One approach is to clone the siRNA cassette into pSOS and then to subcloneGOI. An alternative approach is to clone GOI into pSOS, followed by inserting siRNA cassettes. The GOI can be removed with Pac I digestion, yielding a siRNAexpression vector that expresses GFP as a marker. Further, the pSOS vector also contains MSCV LTRs/packaging signal and confers blasticidin-resistant marker thatcan be used to establish mammalian stable lines. (C) A proposed model of pSOS action. The H1-U6 convergent dual promoter-directed expression of siRNA cassetteswill generate small double-stranded RNA duplexes. These siRNAs specifically target and degrade the chimeric transcripts of GFP and GOI, leading to decrease in GFPsignal. GFP, enhanced green fluorescent protein; GOI, gene of interest; nt, nucleotide; PA, polyadenlylation signal.

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(0.3 μg of pCMV-Wnt3A for HEK-293 cells), and 0.4 μg ofsiRNA expression vectors (e.g., pSOS, or pSOS-siBC-U6)using LipofectAMINE. At the indicated time points, cells werelysed, and subjected to luciferase assays using Promega'sLuciferase Assay Kit. Transfection efficiency was normalizedby β-galactosidase activity. Each assay condition was carriedout in triplicate.

2.6. Immunofluorescence staining

Immunostaining was carried out as described (Luo et al.,2004; Peng et al., 2004). Briefly, subconfluent HEK-293 andHCT116 cells were transfected with pSOS-siBC and controlvectors. At 3 days after transfection, cells were fixed withmethanol at −20 °C for 15 min and washed with PBS. The fixedcells were permeabilized, blocked with 10% goat serum, andfollowed by incubation with anti-β-catenin monoclonal anti-body (Santa Cruz Biotechnology). After washing, the cells wereincubated with anti-mouse IgG secondary antibody labeled withAlexa Fluor 594 (Molecular Probes). The presence of β-cateninprotein was examined under a fluorescence microscope. Stains

without the primary antibody, or with control IgG, were used asnegative controls.

3. Results and discussion

3.1. Development of the pSOS system for selecting optimalsiRNA sites

Although RNAi shows great promise in basic and transla-tional research, selection and validation of effective siRNAtarget sites remain an empirical and time-consuming process. Tosimplify the procedure for selecting optimal siRNA sites, wedeveloped a fluorescence-based screening system, namely thepSOS vector, to identify effective target sites for RNAi. Asillustrated in Fig. 1A, pSOS is a versatile vector and has thefollowing important features. First, the expression of siRNA isdriven by dual convergent H1 and U6 promoters. Oligonucle-otide cassettes, which contain candidate target sites (n19–27)preceded with one extra A at the 5′-end and followed by TTTTat the 3′-end, can be cloned into the Sfi I sites of the pSOSvector (Fig. 1B). In this configuration, both the sense and anti-

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sense strands of siRNA duplex would contain TTTT transcrip-tion terminators at the 3′-ends. Second, a chimeric transcriptcontaining GFP and gene of interest (GOI) is driven by humanElongation Factor 1α (hEF1α) promoter (Fig. 1C). Thus, GFPexpression is directly affected by the knockdown efficacy of thetarget gene by candidate siRNAs (Fig. 1C). We have alsoincluded a stop codon at the end of GFP coding region to ensurethat no GFP fusion proteins would be produced from chimerictranscripts, excluding the possible generation of GFP fusionproteins with diminished fluorescence signal. Third, the linkersite is flanked with Pac I sites so that the target gene can beremoved with Pac I digestion. In this case, GFP expression isrestored and serves as a marker for siRNA gene delivery. Lastly,the pSOS vector is retroviral vector-based and expresses a

Fig. 2. Silencing of GFP expression using pSOS-based siRNA expression vectors. (Aletters; one was a 21nt site, and other two were 27nt sites) were tested. A mutantnucleotides are underlined. (B) RNAi-mediated inhibition of GFP expression. HEplasmids, whose sense-strand expression was driven by either H1 (e.g., siGFP1H) oroff-target control. Expression of GFP was recorded at 5 days after transfection under aof the three GFP target sites. HEK-293 cells were transfected with an equal amountpoints, cells were lyzed and subjected to fluorescence measurement (mean±SD). Trcondition was done in triplicate. Representative results of three independent experim

blasticidin-resistant marker, so the resultant vectors can be usedto establish stable lines expressing siRNAs.

3.2. Silencing of GFP expression using the pSOS system

We first sought to test whether the pSOS system couldeffectively express siRNAs from the convergent dual H1 andU6 promoters. We designed three pairs of oligo cassettescorresponding to three target sites of GFP coding region:siGFP1 contained 21 nucleotides, siGFP2 and siGFP3contained 27 nucleotides, respectively (Fig. 2A). These siteshave been reported as effective siRNA sites of GFP (Tran et al.,2003; Kaykas and Moon, 2004; Kim et al., 2005). As a control,we introduced four mutant nucleotides into the targeting

) Candidate siRNA sites of the GFP coding region. Three target sites (in capitalsiGFP1 site was used to determine possible off-site targeting activity. MutantK-293 cells were transfected with an equal amount of pSOS, or pSOS-siGFPU6 (e.g., siGFP1U) promoter. The siGFP1 mutant (i.e., siGFP1mU) was used asfluorescence microscope. (C) Fluorescence measurement of silencing efficiencyof pSOS, or pSOS-siGFP plasmids, along with pCMVβ. At the indicated timeansfection efficiency was normalized with β-galactosidase activity. Each assayents are shown.

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sequence of siGFP1, resulting in siGFP1m. The wild type andmutant GFP siRNA cassettes were subcloned into the Sfi I sitesof pSOS. For each target site, two constructs, in which thesense-strand was driven by either H1 or U6 promoter, weregenerated because of the identical overhangs in both orienta-tions produced by the Sfi I digestion. For example, the twoconstructs for siGFP1 sites were designated as pSOS-siGFP1Hand pSOS-siGFP1U, respectively. The resultant pSOS vectorsfor the mutant siGFP1 sites were designated as pSOS-siGFP1mH and pSOS-siGFP1mU, respectively.

When the pSOS-siGFP vectors were transfected into HEK-293, GFP expression was readily detected in all samples at 24 hafter transfection. However, at five days after transfection, GFPsignal decreased remarkably in the cells transfected with pSOS-siGFP1 and pSOS-siGFP3 vectors, while cells transfected withpSOS-siGFP2 vectors exhibited a modest but noticeablereduction in GFP signal, when compared with the pSOS control(Fig. 2B). The pSOS vector-mediated knockdown of GFPexpression was seemingly specific, as the mutant siGFP1mvectors did not affect the GFP expression. When the GFPfluorescence was quantitatively measured in time coursestudies, the decrease of GFP signal in pSOS-siGFP-transfectedcells was observed in a time-dependent manner (Fig. 2C). Forexample, siRNAs derived from both siGFP1 and siGFP3exhibited approximately 30% to 60% decrease of GFP signal at24 h after transfection, while at day 7 their GFP signalremarkably decreased to b10% of the pSOS control. The resultswere reproducible in other cell lines, such as HCT116 (data notshown). Interestingly, although there is a sequence overlaybetween siGFP1 and siGFP2, their gene-silencing efficienciesvaried significantly. Our results also demonstrated that both21nt and 27nt target sites can be equally efficient in geneknockdown although a simple extension of a shorter site (e.g.,19 to 21nt) may not be necessarily effective. Our results alsoindicate that siRNAs whose sense-strand expression was drivenby the U6 promoter were more effective than those driven bythe H1 promoter. Nevertheless, our findings demonstrate thatthe expression of siRNAs driven by the convergent H1 and U6dual promoters is effective in gene silencing.

3.3. Selection and validation of siRNA target sites of humanβ-catenin

As a further proof-of-concept, we chose to silence human β-catenin expression. We first cloned the coding region (267nt–2266nt) of human β-catenin coding region (267nt–2266nt) intothe polylinker of pSOS, resulting in pSOS-BC. Seven targetsites were chosen, six of which were selected by using theWhitehead/MIT siRNA Selection (http://jura.wi.mit.edu/bioc/siRNAext/) (i.e., BC-1 to BC-3) and Dharmacon's siDESIGN(http://www.dharmacon.com/) (i.e., BC-4 to BC-6). Site BC-7was previously reported (van de Wetering et al., 2003) (Fig.3A). Oligo cassettes were subcloned into the Sfi I sites of pSOS-BC. For each target site, we selected the constructs whosesense-strand was driven by U6 promoter. When these pSOS-based siRNA vectors targeting human β-catenin were trans-fected into HEK-293 cells, at 5 days after transfection a

significant reduction of GFP signal was observed in BC-2, BC-3, BC-4, BC-5, and BC-6 targeting vectors transfected cells,while BC-1 and BC-7 transfected cells exhibited a modestdecrease in GFP signal intensity (Fig. 3B). Results fromquantitative fluorescence measurements were largely consistentwith that from the fluorescence microscopic detection (Fig. 3C).The siRNAs derived from the target sites of human β-cateninwere shown to effectively inhibit GFP expression in a time-dependent manner (Fig. 3C). These results demonstrate that β-catenin siRNAs can effectively knockdown the chimerictranscript between GFP and human β-catenin.

To convert the pSOS-based vectors from a screening systeminto a convenient expression system, we removed the codingregion of human β-catenin from the pSOS-siBC vectors.According to our working hypothesis outlined in Fig. 1C, theremoval of the target gene should restore GFP expression. Afterthe removal of β-catenin sequence (resulting pSOS-siBC-U6vectors), these vectors were transfected into HEK-293 cells,GFP expression was readily detected at 24 h after transfection(Fig. 4A). GFP expression, in this case, serves as a marker fortransfection. We further analyzed the knockdown of β-cateninexpression by the siBC-U6 vectors. Using immunofluorescencestaining assay, we demonstrated that β-catenin expression wassignificantly inhibited in HEK-293 cells transfected with thesiBC-U6 vectors (Fig. 4B).

3.4. Correlation between the inhibition of GFP signal and theknockdown of β-catenin expression and its signaling activity bycandidate siRNAs of β-catenin

We next analyzed whether the pSOS-siBC vectors-mediateddecrease in GFP signal correlates with inhibition of β-cateninsignaling. To quantitatively assess β-catenin signaling activity,we used the well-established β-catenin/Tcf luciferase reporter,pTOP-Luc (He et al., 1998a, 1999; Zhou et al., 2003; Si et al.,2006). HEK-293 cells were co-transfected with pSOS-siBCs,pTOP-Luc, pCMV-Wnt3A, and pCMVβ. Transfected cellswere lyzed at 3 and 6 days and subjected to luciferase assay. Asshown in Fig. 4C, the decreases in β-catenin/Tcf4 reporteractivity in general correlate well with the GFP expression levelsshown in Fig. 3C. Interestingly, BC-7U6 exhibited a morepronounced inhibition of Tcf4 reporter activity (i.e., 70–80%inhibition) than that of GFP expression (i.e., 20–30%inhibition). Although the reason is not completely known, itmay be due to the different silencing efficiency of siBC7 of theGFP-β-catenin chimeric mRNA vs. the endogenous β-cateninmRNA. It has been well known that siRNA silencing efficacy issignificantly affected by the secondary structure of the targetedtranscripts. Nonetheless, we further demonstrated that siBC7was shown to knockdown β-catenin expression, as effectivelyas siBC4, at mRNA level using qPCR (Supplementary Fig. 1Cand D). Overall, our results suggest that GFP expression may beused as a surrogate marker of gene-silencing efficiencymediated by candidate siRNAs of a target gene. Similar resultswere obtained in the human colon cancer line HCT116, whichharbors an oncogenic mutation of β-catenin and exhibitsconstitutively active β-catenin/Tcf4 signaling (Morin et al.,

Fig. 3. Selection and validation of siRNA target sites of human β-catenin using pSOS-based vectors. (A) Candidate target sites of human β-catenin coding region(267nt–2266nt). Seven target sites were chosen by using the Whitehead/MIT siRNA Selection (http://jura.wi.mit.edu/bioc/siRNAext/) (i.e., BC-1 to BC-3) andDharmacon's siDESIGN (http://www.dharmacon.com/) (i.e., BC-4 to BC-6), and from a published study (BC-7) (van de Wetering et al., 2003). (B) Inhibition of GFPexpression by siRNAs targeting human β-catenin. Subconfluent HEK-293 cells were transfected with an equal amount of pSOS-BC, or pSOS-siBC plasmids. GFPexpression was recorded at 5 days under a fluorescence microscope. Representative results of three independent experiments are shown. (C) Quantitative comparisonof the knockdown efficiency of GFP signal by pSOS-based siRNAvectors targeting human β-catenin. HEK-293 cells were transfected with an equal amount of pSOS-BC, or pSOS-siBC plasmids, along with pCMVβ. At the indicated time points, the transfected cells were lyzed and subjected to fluorescence measurement (mean±SD). Transfection efficiency was normalized with β-galactosidase activity. Each assay condition was done in triplicate.

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1997). The introduction of the pSOS-siBC-U6 vectors intoHCT116 cells remarkably decreased the overall expression andnuclear accumulation of β-catenin (data not shown), and thesiBC-U6 vectors were shown to inhibit approximately 90% ofthe β-catenin/Tcf4 reporter activity in HCT116 cells (Fig. 4D).

This magnitude of inhibition should be considered significantgiven the fact that HCT116 cells have constitutively active β-catenin/Tcf4 signaling. Taken together, our results have proventhe principle of the pSOS-based system for selection andvalidation of siRNA target sites.

Fig. 4. Correlation between siBC silencing efficacy and inhibition of Wnt/β-catenin signaling activity. (A) Restoration of GFP expression of pSOS-siBC vectors. Theinitial pSOS-based siBC vectors (i.e., U6-driven sense-strand vectors) were cut with Pac I and re-ligated to remove human β-catenin. The resulting siBC-U6 vectorswere transfected into subconfluent HEK-293 cells. At 24 h, GFP expression was recorded under a fluorescence microscope. (B) Correlation between siRNA silencingefficiency and inhibition of β-catenin expression in HEK-293 cells. HEK-293 cells were co-transfected with pSOS-siBC-U6 vectors or pSOS, and pCMV-Wnt3A. At3 days, cells were fixed and subjected to immunofluorescence staining using an anti-β-catenin antibody, as described inMaterials and methods Section. (C) Correlationbetween siRNA knockdown efficiency and inhibition of β-catenin signaling activity in HEK-293 cells. Subconfluent HEK-293 cells were co-transfected with pSOS-siBC-U6 vectors, pTOP-Luc, pCMV-Want3A, and pCMVβ. At the indicated time points, cells were lyzed and subjected to luciferase assay (mean±SD). Transfectionefficiency was normalized with β-galactosidase activity. Each assay condition was done in triplicate. (D) Correlation between siRNA silencing efficiency andinhibition of β-catenin expression in HCT116 cells. Subconfluent HCT116 cells were co-transfected with pSOS-siBC-U6 vectors, pTOP-Luc, and pCMVβ. At theindicated time points, cells were lyzed and subjected to luciferase assay (mean±SD). Transfection efficiency was normalized with β-galactosidase activity. Each assaycondition was done in triplicate.

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3.5. Knockdown mediated by pSOS-based retroviral andadenoviral vectors

To further facilitate efficient delivery of the validatedsiRNAs in vitro and in vivo, we subcloned the H1 and U6convergent dual-promoter system into a recombinant adenoviralshuttle vector expressing siRNAs, resulting in pSES (Fig. 5Band www.boneandcancer.org/resources.htm), which is compat-ible with our previously developed AdEasy system (He et al.,1998b). Thus, once target sites are validated, the same oligocassettes can be subcloned into pSES vector and recombinantadenoviruses expressing siRNAs will be subsequently generat-ed by using the AdEasy system.

As proof-of-principle experiments, we tested both retroviral(Fig. 5A) and adenoviral vectors to express the siRNA cassettesselected from the above studies. As shown in Fig. 5C and D, astable pool of 293 cells established using the retroviral vectorpSOS-siBC5U6 expressed GFP, and reduced β-catenin expres-

sion by approximately 70%, when compared with 293-SOScontrol pool using quantitative real-time PCR. Accordingly, weconstructed two adenoviral vectors expressing siBC5 andsiBC6. As shown in Fig. 5E and F, the adenoviral vectorseffectively transduced human osteosarcoma MG63 cells, andinhibited the endogenous β-catenin expression, as assessed byquantitative real-time PCR. It is conceivable that the pSOSsystem can be further configured into lentiviral and/or AAVvectors.

3.6. The pSOS is a versatile and user-friendly gene-knockdownsystem

In the reported pSOS system, we chose to use GFP as areporter for gene silencing. The advantage of using fluores-cence-based analysis is to allow us to assess gene-knockdownefficiency in live cells. However, the fluorescence microscopicdetection is not quantitative, although the fluorescence signal

Fig. 5. Viral vector-mediated knockdown of β-catenin expression. Schematic representation of the retroviral vector pSOS (A) and adenoviral shuttle vector pSES (B),which are used to generate viruses expressing siRNA target sites. (C) GFP expression of the retroviral vector pSOS or pSOS-siBC5U6-mediated 293 stable cells. (D)Quantitative real-time PCR analysis of β-catenin expression in 293-SOS and 293-siBC stable lines. (E) Effective transduction of human osteosarcoma MG63 cells byadenoviruses expressing siBC5U6 and siBC6U6 target sites. Subconfluent MG63 cells were infected with the same titer of AdsiBC5 or AdsiBC6. Expression of RFPwas recorded at 30 h after infection using a fluorescence microscope. (F) Adenovirus-mediated knockdown of β-catenin in MG63 cells. Subconfluent MG63 cells wereinfected with the same titer of AdsiBC5, AdsiBC6, or AdRFP control virus. At 48 h after infection, total RNAwas collected, subjected to reverse transcription reactionsand quantitative real-time PCR analysis of β-catenin expression. Representative data from two independent experiments are shown (mean±SD). For the quantitativereal-time PCR analyses, all samples were normalized with endogenous GAPDH. Each assay condition was done in triplicate.

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can be analyzed quantitatively using a fluorometer. It isconceivable that other reporters, such as luciferase, SEAP orβ-galactosidase, can be used in this system. We also observedno interferon induction when the pSOS-based siRNA expres-sion vectors were introduced into mammalian cells (Supple-mentary Fig. 2). Our results also indicate that there is atranscriptional bias for the commonly-used H1 and U6 Pol IIIpromoters, as U6-driven sense-strand vectors exhibited a more

potent silencing effect than that of the H1-driven ones, althoughthis bias is seemingly site-dependent (Supplementary Fig. 1Aand B). Interestingly, the same dual promoters have been usedin several studies and none of them has reported suchphenomenon (Tran et al., 2003; Kaykas and Moon, 2004;Zheng et al., 2004; Chen et al., 2005; Seyhan et al., 2005).Currently, we do not have any satisfactory explanations for thisgene-silencing bias mediated by H1 vs. U6 promoters.

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Nonetheless, it has been recently reported that U6 promoter ismore efficient than H1 promoter in gene silencing both in vitroand in vivo (Makinen et al., 2006).

In summary, we have developed a fluorescence-basedsiRNA screening method and demonstrated its utility forevaluating the gene-knockdown efficiency of candidate siRNAsites. The GFP-based assay for gene-silencing efficiency can bequalitative and quantitative. Reduction of GFP signal is closelycorrelated to knockdown efficiency of the expression andfunctional activity of target genes. Thus, the pSOS system is anefficient, versatile, and yet user-friendly tool for selecting,validating, and delivering optimal siRNA sites for RNAi-mediated gene silencing.

Acknowledgements

The authors thank Drs. David Engelke of University ofMichigan, Ann Arbor, MI, and Hans Clevers of NetherlandsInstitute for Developmental Biology, Utrecht, The Netherlands,for providing pTZU6+ 1 and pTER vectors, respectively. Thiswork was supported in part by research grants from theNatural Science Foundation of China (QL, #30500602; QK,#30600625), The Brinson Foundation (TCH), OREF (RCH),American Cancer Society (TCH & HHL), and the NIH (RCH &TCH). TCH was a recipient of the Outstanding Overseas YoungInvestigator Collaboration Award from the Natural ScienceFoundation of China (NSFC #30228026) and a recipient of theBayu Scholar of Chongqing Municipality, Chongqing, China.Vector maps and sequences for pSOS and pSES are available at:www.boneandcancer.org/resources.htm. Request for pSOS andpSES vectors should be directed to tche@surgery.bsd.uchicago.edu.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.gene.2007.02.030.

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