Gene 558 (2015) 278–286

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Methods paper

A novel siRNA validation system for functional screening of effective RNAi targets in mammalian cells and development of a derivative lentivirus delivery system Gang Huang a,d,1, Qiangguo Gao b,1, Yongliang Zhao c, Zongming Dong c, Tengfei Li c, Xinyin Guan a, Jianxin Jiang d,⁎ a

Department of Medical Genetics, College of Basic Medicine, Third Military Medical University, Chongqing 400038, China Department of Cell Biology, College of Basic Medicine, Third Military Medical University, Chongqing 400038, China c Department of General Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China d State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China b

a r t i c l e

i n f o

Article history: Received 24 September 2013 Received in revised form 17 December 2014 Accepted 25 December 2014 Available online 28 December 2014 Keywords: siRNA screening Dual luciferase assay Lentivirus delivery

a b s t r a c t RNA interference technology is a widely used tool for the regulation of gene expression at the posttranscriptional level. One major challenge is to find the effective short interfering (si)RNA for target gene rapidly and easily, and then to deliver the siRNA into cells or tissues with high efficiency. Here, we designed a novel siRNA validation vector using a dual luciferase reporter system for the functional screening of effective RNAi targets in mammalian cells. Then, based on a siRNA expression cassette, we developed a derivative lentivirus delivery system to infect the appropriate cells or tissues for the efficient knockdown of target gene expression. Based on this system, we used human IRF7 gene, a key regulatory factor for the differentiation of monocytes to macrophages, as an example. We screened for the optimal siRNA, then packaged it into a lentiviral siRNA expression system. Then, monocytes were infected and we confirmed the knockdown of IRF7 expression could inhibit the differentiation of monocytes to macrophages. To validate our method further, we also screened and identified optimal siRNA for human TLR4 gene. In summary, we developed a novel siRNA validation system to identify optimal siRNA to target genes rapidly. In addition, the lentivirus system is an efficient tool for siRNA delivery for the further study of target gene function. Taken together, this represents an efficient and user-friendly strategy to validate and deliver siRNAs. © 2015 Elsevier B.V. All rights reserved.

1. Introduction RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discovery (Chen and Xie, 2012; Guzman-Villanueva et al., 2012). One major challenge of using RNAi is to identify the effective short interfering RNA (siRNA) target sites of a given gene and transport it efficiently to the appropriate cell or tissue to knockdown the expression of target gene (Chen and Xie, 2012; Guzman-Villanueva et al., 2012; Mohr and Perrimon, 2012). Although several published bioinformatic prediction models (Stormo, 2006; Tilesi et al., 2009) and some fluorescencebased siRNA sequence selection systems (Luo et al., 2007; Zheng et al., 2011) have proven useful, the process to select and validate optimal Abbreviations: dsRNA, double-stranded RNA; hRluc, humanized Renilla luciferase; IRF7, interferon regulatory factor 7; NC, negative control; PGK, phosphoglycerate kinase; psiSDR, plasmid for siRNA screening with dual reporters; RNAi, RNA interference; siRNAs, short interfering RNAs; SOE, splicing by overlapping extension; TLR4, Toll like receptor 4. ⁎ Corresponding author. E-mail address: [email protected] (J. Jiang). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.gene.2014.12.063 0378-1119/© 2015 Elsevier B.V. All rights reserved.

siRNA sites for a given gene remains empirical and laborious. In addition, introducing siRNAs or expression vectors to primary cells or hard-transfected cell lines such as lymphocytes and PC-12 cells with a high efficiency is still a recognized problem (Aagaard and Rossi, 2007). Here, we developed a reliable and quantitative reporter-based siRNA validation system for the functional screening and delivery of effective RNAi probes in mammalian cells. It is a dual luciferase assay-based selection system, named psiSDR (plasmid for siRNA screening with dual reporters), and is based on the premise that candidate siRNAs would knockdown the chimeric transcript of luciferase and target genes. The expression of siRNA was driven by the opposing convergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that relative luciferase activity signal reduction was closely correlated with siRNA knockdown efficiency of human interferon regulatory factor 7 (IRF7). It was then packaged into a lentiviral siRNA expression system and used to infect primary monocytes and knockdown of hIRF7 expression inhibited the differentiation of monocytes to macrophages. In addition, we repeated our experiments on human TLR4 (Toll like receptor 4) gene, then validated the efficiency and wide applicability of this system. The

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use of psiSDR and the lentivirus delivery system should facilitate the selection and validation of candidate siRNA sites and provide efficient tools for delivering siRNAs to mammalian cells via lentiviral vectors. Thus, the psiSDR and corresponding lentivirus delivery system provides a powerful strategy to validate and deliver highly effective siRNAs. 2. Materials and methods 2.1. Cell culture and reagents Primary human monocytes were obtained from healthy donors with written informed consent and this study was approved by the Medical Ethics Committees of the Third Military Medical University. The study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki. Fresh whole blood was drawn into vacutainer tubes (Becton Dickinson & Co., Franklin Lakes, NJ, USA) containing EDTA. Peripheral blood mononuclear cells (PBMCs) were isolated using FicollHypaque (TBD, Tianjin, China). CD14+ monocytes were isolated from PBMCs by negative selection using Human Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's instructions. Monocyte purity was verified as N 95% by anti-CD14 staining (Cat. #17-0149; eBioscience, San Diego, CA, USA) using flow cytometry. Monocytes were seeded at 1 × 106 cells/well in 12-well plates (Corning Incorporated, Corning, NY, USA) and maintained in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat inactivated human AB serum (Sigma, Batavia, IL, USA). Macrophage differentiation was induced by treatment with 20 ng/ml macrophage colony-stimulating factor (M-CSF, BD Biosciences, San Jose, CA, USA) for 7 days (D'Onofrio and Paradisi, 1983). Every third day, half of the medium was removed and replaced with fresh complete nutrient medium. All monocyte-to-macrophage differentiation experiments were performed in 10% human serum plus 20 ng/ml M-CSF, unless otherwise stated. HEK-293T and COS7 cell lines were purchased from ATCC (ATCC, Rockville, MD, USA). These cells were maintained in complete DMEM containing 10% FBS (Gibco BRL). Unless otherwise indicated, all chemicals were purchased from Sangon Biological Engineering Technology Company (Sangon, Shanghai, China). Restriction endonucleases and T4 DNA ligase were purchased from NEB (New England Biolabs, Ipswich, MA, USA), and plasmid DNA miniprep kits and agarose gel DNA fragment recovery kit were from Omega (Omega Bio-Tek, Norcross, GA, USA). Lipofectamine 2000 was purchased from Invitrogen (Grand Island, NY, USA). The DNA templates for the IRF7 gene or TLR4 gene siRNAs and PCR primers were synthesized by Sangon. DNA sequencing was conducted by Sangon. 2.2. Design and construction of a novel siRNA validation plasmid psiSDR Based on the template of plasmid pmirGLO (Promega, Madison, WI, USA), we designed and synthesized three pairs of primers (Table 1) to clone the DNA fragments for luc2 reporter gene, human phosphoglycerate kinase (PGK) promoter and humanized Renilla luciferase (hRluc)

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reporter gene, respectively. According to the manufacturer's instructions, PCR was performed under the following conditions: plasmid DNA template was denatured at 99 °C for 10 min, followed by 35 cycles of amplification (98 °C for 20 s, 68 °C for 2 min) and 8 min at 72 °C. The anticipated size of PCR products was identified, separated and recovered by electrophoresis using a 1% agarose gel. Based on the 16 nucleotide (nt) complementary overlapping sequence between these three templates (shown in Table 1 as underlined), splicing by overlapping extension (SOE)-PCR (Vallejo et al., 2008) was used to obtain the fused DNA of luc2, PGK promoter and hRluc tandemly. The fused DNA was amplified using primers P1 and P6. PCR was performed as follows: 98 °C for 6 min, then 30 cycles of 98 °C for 20 s and 68 °C for 3 min, followed by 68 °C for 10 min. The anticipated PCR product size recovered was 3681 base pairs (bp). Green fluorescent protein (GFP)-based mammalian siRNA screening vector pSOS-HUS (a kind gift from Dr. Luo Qing, Pediatrics Institution of Chongqing Medical University, Chongqing, China) was digested with Eco47 III (blunt end) and Bgl II, while the 3681 bp fused DNA was digested with BamH I (BamH I has a compatible end with Bgl II) only. After digestion, the fused DNA and the linearized pSOS-HUS vector were recovered, ligated and transformed into Escherichia coli DH10B. Transformed positive clones for psiSDR were screened by colony PCR using primers P3 and P4, and further verified by sequencing (Fig. 1). 2.3. Construction of the double-cloned psiSDR-hIRF7i plasmids for screening optimal siRNA Recombinant hIRF7/pTA2 plasmid containing the human IRF7 intact open reading frame (GenBank ID: U53830.1) was digested with EcoR V and Xba I, and the siRNA screening plasmid psiSDR was digested with Pme I and Nhe I. After digestion, the hIRF7 cDNA (1593 bp) and the linearized psiSDR vector were recovered, ligated using T4 DNA ligase, and transformed into E. coli DH10B. Transformed positive clones for psiSDR-hIRF7 were screened by colony PCR using primers P7 and P8, and further verified by EcoR I digestion (5.9/2.2/1.3 kb). Then psiSDR-hIRF7 vector was digested with Sfi I and recovered for the next ligation. siRNA sequences targeting hIRF7 (GenBank ID: U53830.1) were selected using standard siRNA design algorithms (Moore et al., 2010; Tafer, 2014). We designed three pairs of siRNA template oligonucleotides targeted against hIRF7 gene expression, as shown in Table 2. Eight oligonucleotides were thus synthesized. Five microliters of the complementary oligonucleotides (100 μM) was mixed in a 1.5-ml Eppendorf tube with 4 μl 5× annealing buffer (Beyotime, HaiMen, Jiangsu, China) and distilled water, in a total volume of 20 μl. The Eppendorf tube was place on a 99 °C heat block for 10 min, and then removed and allowed to cool at room temperature. The Eppendorf tube was briefly centrifuged at full speed to recover the reaction solution, which was stored on ice or at 4 °C until ready for use. The construction procedures used standard molecular cloning techniques. Briefly, an annealed oligonucleotide duplex was inserted into the Sfi I restriction enzyme site in

Table 1 Primer sequences for SOE-PCR and cloning. Designation

Primers sequence

Tm

Amplicon size (bp)

luc2 reporter gene

P1: (F) 5′-GCTTGGCAATCCGGTACTGTTGGTAA-3′ P2: (R) 5′-TTGTACGCTTTACCACATTTGTAGAGGTTTTACTTGCT-3′ P3: (F) 5′-GTGGTAAAGCGTACAATTAAGGGATTATGGTA-3′ P4: (R) 5′-GAAGAATCTGGGCTGCAGGTCGAAAGGCC-3′ P5: (F) 5′-GCAGCCCAGATTCTTCTGACACAACAGTC-3′ P6: (R) 5′-CGGGATCCTTACTGCTCGTTCTTCAGCAC-3′ (Xba I site) P7: (F) 5′-GGAATTCACCTGACCGCCACCTAACTG-3′ P8: (R) 5′-GCTCTAGAGTTCTCATTAGACTGGGTTCTAG-3′ P9: (F) 5′-CGGGATCCAGCTTAATTCGAACGCTGACGT-3′ (BamH I site) P10: (R) 5′- CCCTCGAGCAAGGTCGGGCAGGAAGAG-3′ (Xho I site)

68 °C

2076

68 °C

641

68 °C

996

68 °C

1583

68 °C

566

PGK promoter hRluc reporter gene hIRF7 cDNA siRNA expression cassette

SOE, splicing by overlapping extension; hIRF7, human interferon regulatory factor 7; siRNA, short interfering RNA; PGK, phosphoglycerate kinase; hRluc, humanized Renilla luciferase.

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2.4. Transfection and dual luciferase reporter assay for screening optimal hIRF7 siRNA Double-cloned psiSDR-hIRF7i plasmids were transfected into HEK293T or COS7 cells. The cells were plated into 12-well plates and transfected with 1 μg miniprep purified sterile plasmid DNA (per well) using Lipofectamine 2000 in duplicate, according to the manufacturer's instructions. After 72 h, the cells were harvested and lysed for luciferase assay using a dual luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Renilla luciferase was used for normalization. 2.5. Production of derivative lentiviral virions for optimal hIRF7 siRNA

Fig. 1. A novel vector system for the rapid screening of effective siRNA target sites. (A) Construction of the psiSDR vector. The length of the vector is approximately 7.9 kb. The novel vector is composed of five elements, including a Luc2-Flag or hRluc-Flag expression cassette, multiple cloning site 1 (MCS1) or MCS2 for the gene of choice (GOC) cloning, siRNA expression cassette and kanamycin/neomycin-resistant expression cassette. There is a stop codon between the Luc2-flag fusion protein and MCS1 cloning site and another stop codon between the hRluc-flag fusion protein and MCS2 cloning site. Therefore, both cassettes can produce a Luc2/hRluc-Flag-GOC chimeric transcript but not generate Luc2/hRluc-Flag-GOC fusion proteins. (B) Unique restriction sites in the MCS1 and MCS2 of psiSDR vector.

the siRNA screening psiSDR-hIRF7i vector using T4 DNA ligase. We prepared a control ligation mixture in a separate 200-μl Eppendorf tube by replacing the annealed oligonucleotide duplex with distilled water. After transformation of E. coli DH10B, the sample plate should have at least 10-fold more colonies than the control plate for a successful digestion–ligation–transformation experiment (Lu and Zhu, 2009). Three recombinant siRNA screening psiSDR-hIRF7i clones per plate were picked and verified by sequencing (Fig. 2). These were named psiSDR-hIRF7-i1, i2, i3 and NC (negative control).

Table 2 siRNA target sequences for hIRF7 gene and for hTLR4 gene. Designation

Primers sequence

Target location

hIRF7-i1

i1-F: 5′-aACGACATCGAGTGCTTCCTTATtttt-3′ i1-R: 5′-aATAAGGAAGCACTCGATGTCGTtttt-3′ i2-F: 5′-aCGGAGAGTGGCTCCTTGGAGAGtttt-3′ i2-R: 5′-aCTCTCCAAGGAGCCACTCTCCGtttt-3′ i3-F: 5′-aGGCAGATCCAGTCCCAACCAAGtttt-3′ i3-R: 5′-aCTTGGTTGGGACTGGATCTGCCtttt-3′ NC-F: 5′-aCAACAAGATGAAGAGCACCAAtttt-3′ NC-R: 5′-aTTGGTGCTCTTCATCTTGTTGtttt-3′ i1-F: 5′-aCTGCGTGGAGGTGGTTCCTAATtttt-3′ i1-R: 5′-aATTAGGAACCACCTCCACGCAG tttt-3′ i2-F: 5′-aTGGCCTTCCTCTCCTGCGTGAGtttt-3′ i2-R: 5′-aCTCACGCAGGAGAGGAAGGCCAtttt-3′ i3-F: 5′-aACCTCTCTACCTTAATATTGACtttt-3′ i3-R: 5′-aGTCAATATTAAGGTAGAGAGGTtttt-3′

nt 1761–1782 (U53830.1) nt 334–355 (U53830.1) nt 898–919 (U53830.1)

hIRF7-i2 hIRF7-i3 siRNA negative control hTLR4-i1 hTLR4-i2 hTLR4-i3

nt 382–403 (NM_138554.4) nt 342–363 (NM_138554.4) nt 606–627 (NM_138554.4)

hIRF7, human interferon regulatory factor 7; hTLR4, human Toll like receptor 4; NC, negative control; siRNA, short interfering RNA; nt, nucleotide.

Based on the modified promoterless pLenti7.3/V5-TOPO lentiviral vector (Invitrogen), we prepared derivative lentivirus delivery vectors for optimal siRNA of hIRF7. First, the siRNA expression cassettes (H1 promoter, Insert and U6 promoter) for hIRF7-i1 and NC were amplified using primers P9 and P10 (Table 1). PCR conditions were described in 2.2. Then the purified PCR products and the promoterless pLenti7.3/ V5-TOPO plasmid (Fig. 3A) were digested by BamH I and Xho I. After recovery, these were ligated and transformed. Transformed positive clones were screened by colony PCR using primers P9 and P10, and further verified by sequencing. Two siRNA constructs for hIRF7 knockdown were named LV-siRNA-hIRF7 and LV-siRNA-NC. The lentiviral packaging plasmid psPAX2 and envelope plasmid pMD2G were obtained from Dr. Didier Trono (School of Life Sciences, Lausanne, Switzerland). To prepare virus stocks, 293FT cells (Invitrogen) were co-transfected with siRNA constructs, together with psPAX2 and pMD2G constructs (4:3:2 quality ratio, respectively), using Lipofectamine 2000. The medium was changed after 12 h, and virion-containing medium was collected after 72 h. The viral stocks were centrifuged and filtered through a 0.45-μm filter to remove nonadherent 293FT cells. Then lentiviral virion concentration was performed with Lenti-X™ Concentrator Kit (Cat. #631232; Clontech Laboratories, Mountain View, CA, USA). 2.6. Determining lentiviral titers via flow cytometry for GFP expression The day before transduction, HT-1080 cells were trypsinized and counted. They were then plated in a 6-well plate (2 × 105 cells per well) such that they would be 30–50% confluent at the time of transduction. On the day of transduction, the cells were incubated with serial dilutions (0, 10− 1, 10− 2, 10− 3, 10− 4) of viral stocks plus 8 μg/ml polybrene (Sigma). The following day, the media containing virus was removed and replaced with 2 ml of complete DMEM medium. Five days post-infection, cells were detached, fixed and analyzed by fluorescence-activated cell sorting (FACS) for GFP fluorescence versus number of cells. The percentage of GFP-expressing cells was measured by placing a marker discriminating between GFP-negative and GFPpositive cells. Titration (Gatlin et al., 2003; Giry-Laterriere et al., 2011) was determined by applying the following formula: Titer (HT-1080TU/ml) = 100,000 (target HT-1080 cells) × (% of GFP-positive cells / 100) / volume of supernatant (in ml). 2.7. Transduction of human primary monocytes Briefly, 5 × 105 monocytes per well were plated into a 6-well plate with 2 ml of fresh complete RPMI 1640 medium under the desired cytokine conditions. Then, 0.1 ml of virion stock (about 5 × 108 TU/ml, multiplicity of infection (MOI) = 20) plus 8 μg/ml polybrene (Sigma) was added and incubated at 37 °C for 4 h. A MOI of about 20 is generally used to obtain transduction efficiency of N80%. Monocytes were then washed and cultured as per the conditions required for further differentiation of monocytes to macrophages (D'Onofrio and Paradisi, 1983). After 5 days, the percentage of GFP-positive (GFP +) cells

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Fig. 2. Screening of effective siRNA target sequences for hIRF7. The coding region of hIRF7 gene with a length of approximately 1.6 kb was cloned into MCS1 of psiSDR. Then, resultant plasmids were ligated with chemically synthesized target-specific oligos. The obtained plasmids were termed psiSDR-hIRF7-x(i1–i3, or NC [negative control]). (A) Sequencing results of psiSDR-hIRF7i. The original psiSDR DNA sequence between the U6 promoter and H1 promoter was replaced with hIRF7 siRNA target sites or nonsense siRNA control (underlined in red), respectively. (B) Screening of optimal siRNA target sequences against hIRF7 by dual luciferase assays. Double-cloned psiSDR-hIRF7i plasmids were transfected into HEK-293T or COS7 cells. After 72 h, the cells were harvested and lysed for luciferase assay. Renilla luciferase was used for normalization. The relative luciferase activity in both background cells transfected with the hIRF7-i1-containing double-cloned plasmid was the lowest. *, p b 0.05; **, p b 0.01.

was determined by calculating the number of GFP + cells and total cells from randomly selected microscopic fields under a fluorescence microscope (Leica Microscope DMIRB, Wetzlar, Germany). A total of three microscopic fields, containing at least 100 cells each, were counted for each transduction efficiency test. The knockdown efficiency of hIRF7 was assessed 7 days PI by western blot assay. 2.8. Western blot analysis At 7 days PI, cells were collected and protein was extracted for western blot detection of the target gene product for comparison with the non-infected, LV-siRNA-NC lentivirus infected and LVsiRNA-hIRF7 lentivirus infected groups. Proteins were isolated in radioimmunoprecipitation assay buffer (RIPA, Cat. #P0013B; Beyotime) containing a protease and phosphatase inhibitor cocktail (Cat. #78440; Thermo Fisher Scientific, Rockford, IL, USA). SDS-PAGE was performed in a 12% polyacrylamide gel and proteins were transferred onto a polyvinylidene fluoride membrane in transfer buffer for 1 h using a

Bio-Rad Semi-Dry apparatus. Washes and incubations were performed using standard procedures. Anti-human IRF7 monoclonal antibody (Cat. #AB70069, 1:1000; Abcam, Cambridge, UK) was used as the primary antibody. The secondary antibody was horseradish peroxidase (HRP)-labeled anti-mouse-IgG (1:2000; Zhongshan Bio-tech, Beijing, China). An HRP-labeled anti-GAPDH monoclonal antibody (Clone KC-5G5; 1:5000; KangChen Bio-Tech, Shanghai, China) was used to detect GAPDH as an internal control. Western blots were visualized using ECL reagents (Pierce Biotechnology, USA) and a Storm 860 PhosphorImager. For Figs. 3B and 4C, blots were scanned and quantified using ImageJ software, using GAPDH as loading control. 2.9. Flow cytometry 2.9.1. Apoptosis detection by annexin V/PI staining At 7 days PI, all three group cells were collected, then an APCannexin V apoptosis detection kit (Cat. #88-8007-72, eBioscience) was used to stain for annexin V and phosphatidylinositol (Pradelli et al.)

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Fig. 3. hIRF7 siRNA lentiviral transduction and its effect on the differentiation of monocytes to macrophages. (A) Plasmid map of the modified promoterless pLenti7.3/V5 lentiviral shuffle vector. (B) Representative western blot for evaluation of hIRF7 protein levels after siRNA (negative control (NC) or hIRF7-directed) lentivirus transduction or no lentivirus transduction of primary human monocytes. M-CSF was added to cells after 4 h of lentivirus transduction and incubated for 7 d. GAPDH was used as control. The graph represents quantification of three experiments. *, p b 0.05. (C) Monocytes were transduced with no siRNA (gray, no lentivirus), control siRNA (blue), or hIRF7 siRNAs (red). At 7 d, infected monocytes were stained with PECD71 plus APC-CD14 and analyzed by flow cytometry. (D) Following infection after 7 d, cells were stained with APC-annexin V and PI to detect apoptotic cells and flow cytometric analysis was performed. Results (B to D) are representative of one of three independent experiments from three different donors (n = 3). M-CSF, macrophage colony-stimulating factor.

according to the manufacturer's instructions. Flow cytometry was performed with a FACSCalibur system (BD Biosciences) and data were analyzed with FlowJo Data Analysis Software ver.7.6.1. 2.9.2. Characterization of monocyte-to-macrophage differentiation At 7 days PI, monocyte-to-macrophage differentiation staining was performed on collected cells fixed in 2% paraformaldehyde at room temperature for 10 min. Cells were collected and incubated in blocking solution (PBS plus 5% BSA [Thermo Fisher Scientific], 5% goat serum [Zhongshan Bio-Tech], and human FcR block [Cat. #130-059-901; Miltenyi Biotec]), followed by staining with anti-CD71-PE (Cat. #120719; eBioscience) and anti-CD14-APC (Cat. #17-0149; eBioscience) in blocking solution. For each experiment, appropriate isotype control mAbs were used. Labeled cells were then analyzed by FACS using a FACSCalibur system (BD Biosciences). Results were expressed as a percentage of positive cells. 2.10. Screening and identifying optimal siRNA for human TLR4 gene To further validate the efficiency and wide applicability of this system, another typical gene, i.e. the human TLR4 gene (GenBank ID: NM_138554.4), was chosen as a target gene. We designed three pairs of siRNA template oligonucleotides targeted against hTLR4 gene expression (Table 2). Recombinant hTLR4/pTA2 plasmid containing human TLR4 intact ORF (NM_138554.4) and plasmid psiSDR were digested

with SacI and SalI (TOYOBO, Japan). After digestion, the hTLR4 cDNA (2610 bp) and the linearized psiSDR vector were recovered, ligated using T4 DNA ligase, and transformed into E. coli DH10B. Transformed positive clones for psiSDR-hTLR4 were confirmed by sequencing. Construction of the double-cloned psiSDR-hTLR4i plasmids, screening for optimal siRNA, lentiviral package and transduction to human monocytes were performed as previously stated. The expression of hTLR4 gene was analyzed by western blotting assay. An anti-human TLR4 polyclonal antibody (AB20102b, 1:1000 diluted) was obtained from Sangon (Sangon, China) and used as the primary antibody. Seven days after lentiviral infection, human macrophages were treated with 100 ng of LPS (E. coli, 0111:B4, Sigma) per ml (black bars) for 20 h at 37 °C. Supernatants were collected for a TNF-α ELISA (F02810, Westang, Shanghai, China). 2.11. Statistical analysis The experimental data were subjected to analysis of variance, where a value of P b 0.05 was considered statistically significant. 3. Results 3.1. Characterization of a novel siRNA validation plasmid psiSDR After DNA fragments were cloned for the luc2 reporter gene, hPGK promoter and hRluc reporter gene, they were seamlessly ligated through

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Fig. 4. Screening of effective siRNA sequences for hTLR4, lentiviral transduction and effect on the TNF-α secretion of LPS treated macrophages. (A) Sequencing results of psiSDR-hTLR4i. The hTLR4 siRNA target sites or nonsense siRNA control were underlined in red. (B) Screening of optimal siRNA target sequences against hTLR4 by dual luciferase assays. Double-cloned psiSDR-hTLR4i plasmids were transfected into HEK-293T cells. After 72 h, the cells were harvested and lysed for dual luciferase assay. (C) Representative western blot for evaluation of hTLR4 protein levels after siRNA (negative control (NC) or hTLR4-directed) lentivirus transduction or no lentivirus transduction of primary human monocytes. M-CSF was added to cells after 4 h of lentivirus transduction and incubated for 7 d. GAPDH was used as control. The graph represents quantification of three experiments. *, p b 0.05. (D) Knockdown effect of hTLR4 on LPS-induced TNF-α levels in human macrophages culture supernatant. Seven days after lentiviral infection, human macrophages were treated with 100 ng of LPS per ml for 20 h at 37 °C. Supernatants were collected for a TNF-α ELISA. Results (C, D) are representative from three independent experiments of three different donors (n = 3). *, p b 0.05.

overlap extension PCR, and used to construct the recombinant plasmid psiSDR (Fig. 1) by classical restriction enzyme digestion, T4 DNA ligase ligation and transformation. The product was then confirmed by sequencing, and no mutations were found. 3.2. Characterization of the double-cloned psiSDR-hIRF7i plasmid and screening for optimal siRNA for hIRF7 The successfully constructed recombinant plasmid psiSDR-hIRF7 was digested with EcoR I. The circular plasmid was cleaved into three fragments as follows: 5.9 kb, 2.2 kb (mainly from psiSDR) and 1.3 kb (mainly from hIRF7 cDNA). The plasmids psiSDR and derivative psiSDR-hIRF7 have two Sfi I restriction sites between the U6 and H1 promoters. This site was lost after Sfi I digestion, and hIRF7i fragments cloned into these two sites did not regain the Sfi I restriction site (Luo et al., 2007). As predicted, EcoR I digestion of psiSDR-hIRF7i resulted in three bands as follows: 5.9 kb, 2.2 kb and 1.3 kb. These results indicated that the double-cloned psiSDR-hIRF7i plasmids contained the hIRF7 gene. To confirm that the psiSDR-hIRF7i plasmids contained the correct

inserted hIRF7 siRNA fragments, each of the four correct double-cloned plasmids was sequenced. The H1 sequencing primer was used to sequence the inserted siRNA fragments. The sequences between the H1 and U6 promoters corresponded to the hIRF7 siRNA sequence (Fig. 2A), confirming that the four double-cloned plasmids were successfully constructed. To screen for optimal siRNA for hIRF7, double-cloned psiSDR-hIRF7i plasmids were transfected into HEK-293T or COS7 cells, then assayed for dual luciferase reporter activity. Tay et al. reported that microRNAs could act on the ORF of the target gene to inhibit its expression (Tay et al., 2008; Zhou et al., 2009). Thus, to minimize the bias impact of different background miRNA profiles on the psiSDR-hIRF7i system, we transfected psiSDR-hIRF7i to two cell lines derived from different species and tissues to perform the dual luciferase assay, with the aim of minimizing the artificial deviation of relative luciferase activity. As shown in Fig. 2B, psiSDR-hIRF7-i1 had minimal relative luciferase activity in both cell lines. Taken together, these results suggest that the siRNA transcribed from hIRF7-i1 has the strongest ability to interfere with hIRF7.

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3.3. Production of high titer derivative lentiviral virions for optimal hIRF7 siRNA After self-inactivating third-generation lentiviral vectors LV-siRNAhIRF7 and LV-siRNA-NC were constructed and verified, they were packaged by transient co-transfection to 293FT cells and concentrated by centrifugation. The titers of derivative lentiviral virions were approximately equivalent to 5.0 × 108 TU/ml. The process took approximately 2 weeks to complete. 3.4. siRNA lentiviral transduction and effect on monocyte-to-macrophage differentiation 3.4.1. Characterization of gene-knockdown efficiency of hIRF7 siRNAexpressing lentivirus in human macrophages At 7 days PI, three groups of cells were harvested and analyzed by western blotting. As shown in Fig. 3B, the expression of hIRF7 protein was effectively suppressed in macrophages following infection with LV-siRNA-hIRF7 but not with LV-siRNA-NC. Western blot analysis suggested that the hIRF7-targeting siRNA successfully silenced the endogenous hIRF7 gene expression in the macrophages. 3.4.2. The effect of IRF7 knockdown on monocyte-to-macrophage differentiation IRF7 is a multifunctional transcription factor (Ning et al., 2011). Besides its roles in IFN-mediated immune responses, it also regulates immune cell differentiation and activation and is required for monocyte differentiation to macrophages (Lu and Pitha, 2001). Here we showed that the knockdown of IRF7 inhibited the differentiation of monocytes to macrophages. First, we examined the apoptotic rates of macrophages derived from monocytes (MDMs) following infection with siRNA lentivirus and found that hIRF7 knockdown did not affect the levels of apoptosis (Fig. 3D). Controls, consisting of uninfected MDMs and NC siRNA lentivirus infected MDMs, exhibited levels of apoptosis similar to those of hIRF7 siRNA lentivirus infected MDMs (Fig. 3D), indicating that during the differentiation of monocyte to macrophages, hIRF7 had no significant effect on apoptosis. Second, we examined whether hIRF7 affected the differentiation of monocytes to macrophages. We found that the knockdown of hIRF7 inhibited the differentiation of monocytes to macrophages, as determined by the decrease in CD71 surface expression (Fig. 3C). Together, these data suggest that hIRF7 plays a key role in regulating the differentiation of monocytes to macrophages. 3.5. Screening and identifying optimal siRNA for human TLR4 gene To further test whether our method could be used to screen for effective siRNA sequences for other genes, the well-known human TLR4 gene was chosen as a typical example. After the double-cloned psiSDR-hTLR4i plasmids were confirmed by sequencing (Fig.4A), they were transfected into HEK-293T cells to screen for optimal siRNA for hTLR4, and then assayed for dual luciferase reporter activity. As shown in Fig. 4B, psiSDR-hTLR4-i1 and psiSDR-hTLR4-i2 had minimal relative luciferase activity. These results suggest that the siRNAs transcribed from hTLR4-i1 or hTLR4-i2 have the strongest ability to interfere with hTLR4 expression. Then LV-siRNA-hTLR4-i1/i2 was constructed and packaged to lentiviral virions, and their titers were about 5.0 × 108 TU/ml. Human primary monocytes were purified, infected by lentiviral virions and differentiated to macrophages induced by M-CSF as before. At 7 days PI, three groups of cells were harvested and analyzed by western blotting. The expression of TLR4 protein was effectively knocked down in macrophages following infection with LV-siRNA1-hTLR4 and LV-siRNA2-hTLR4 but not with LV-siRNA-NC (Fig. 4B). It is known that TNF-α is produced in a large amount by macrophages upon TLR4 activation and supports long-term macrophage survival (Rossol et al., 2011; Lombardo et al., 2007; Li et al., 2011).

Here we show that upon the knockdown of TLR4 by RNAi, the level of TNF-α secretion in macrophages induced by LPS was suppressed obviously. These results further suggest that the optimal siRNA sequences screened by our system are effective. 4. Discussion RNA interference (RNAi) is one of the most exciting discoveries of the past decades in functional genomics (Bernards, 2006). RNAi is rapidly becoming an important method for analyzing gene functions in eukaryotes and holds promise for the development of therapeutic gene silencing. RNAi is a post-transcriptional process triggered by the introduction of small double-stranded RNA (dsRNA), which leads to gene silencing in a sequence-specific manner. These dsRNAs are called siRNAs. They feature high specificity, high efficiency and inheritability. Currently, siRNAs are usually designed based on software analysis or literature study. Different software-designed siRNAs targeting the same gene may have different interference efficiencies (Schubert et al., 2005; Stormo, 2006; Strapps et al., 2010; Tiemann and Rossi, 2009). As a result, multiple target sites of a given gene have to be tested and validated experimentally. The validation of silencing efficacy, which is usually based on screening by western blot, Northern blot or quantitative realtime PCR, is laborious and time consuming. Although few fluorescencebased siRNA sequence selection systems have been reported (Kamio et al., 2010; Luo et al., 2007; Zheng et al., 2011), these methods still have some disadvantages, such as bias in judging the efficiency of siRNA target sites for the sake of autofluorescence for strong background signal noise (Choy et al., 2003). Therefore, if the efficient siRNA could be quickly and easily screened from many siRNAs, it would greatly facilitate the subsequent successful silencing of target gene expression with RNAi. This capability would be especially helpful in the study of novel gene function such as newly discovered long noncoding RNAs. Effective RNA interference experiments comprise two basic steps: 1) finding an effective siRNA target sequence and 2) expressing that target sequence in cells or tissues (Lambeth and Smith, 2013). In this study, we describe a novel vector system that offers an easy and fast method for both steps. The convenient and effective siRNA screening plasmid (psiSDR) used in this study is a vector system developed by our group to express small RNA fragments as RNAi. The psiSDR vectors offer a fast and easy method for finding an effective siRNA target sequence. This vector contains the following three cassettes: (1) a firefly luciferase reporter gene expression cassette followed by a multiple cloning site and SV40 poly(A) for the insertion of a target sequence, which results in a fusion transcript of the target gene and reporter gene, including two independent ORFs; (2) siRNA expression cassette containing a dual PoI III promoter driving in opposite directions (U6 and H1 promoters), which results in a 19–23 nt, double-stranded, small RNA fragment (siRNA). Basing on two kinds of novel standard siRNA design algorithms, namely OligoWalk and RNAxs (Lu and Mathews, 2008; Tafer et al., 2008), we selected three shRNAs targeting different regions within the same gene that had the fewest amounts of BLAST matches, that did not overlap a region of SNP, and that targeted different regions within the gene. Through calculating thermodynamic features of sense– antisense hybridization and analyzing the statistical mechanics of the siRNA-target interaction, the OligoWalk web server is constructed to predict efficient siRNA candidates for a given mRNA sequence. It predicts the free energy changes of oligonucleotides binding to a target RNA. The secondary structures of the oligomer and target mRNA are also considered in the OligoWalk algorithm. Another robust siRNA selection tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility, substantially improves the prediction of highly efficient siRNAs. (3) An internal loading control, hRluc reporter gene, which permits normalization of changes in firefly luciferase expression to the expression of Renilla luciferase, making the system more robust and reproducible. If this 19–23 nt sequence is completely complementary to a fragment of the target mRNA, the target mRNA

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will be silenced based on the RNA interference principle. The silencing effect can be directly monitored by examining the reduction or loss of relative luciferase activity. The psiSDR vector has the following advantages: (1) it is convenient for the rapid screening of effective siRNA target sites by one-step transfection with a single double-cloned psiSDR vector and does not need to be normalized by co-transfection; (2) it saves time, without the use of traditional time-consuming and laborious detection methods for RNAi effects, such as quantitative real-time PCR or western blotting and no waiting for phenotypic changes; (3) it can be used widely, is independent of the natural genes and cell lines, can avoid difficult transfection of cells for the screening of effective siRNA targets, the reactions can be temporarily transferred to other easily transfected cells such as HEK 293 or COS7 cell lines, and can avoid the processing of a toxic genes directly to cells; (4) it uses quantitative detection, suitable for high-throughput screening; and (5) if needed, the reporter genes can be excised, such as ligation after restriction enzyme digestion by Eco47 III and Sma I, it can be used as routine siRNA expression plasmid, and it can be selected with G418 after transfection. However, the psiSDR vector has its own limitations. Two cycles of molecular cloning to insert the target gene and siRNA target sequences one by one is required and this technique requires the researcher to be familiar with molecular biology techniques. Introducing siRNAs or its expression vector to the primary cells or hard-transfected cell lines with high efficiency remains challenging (Aagaard and Rossi, 2007). The lentiviral vector is convenient and inexpensive for high-efficiency transduction of intracellular siRNA expression, making it one of the most widely used biological viral vectors. It has many advantages including a wide host range, low pathogenicity for humans and effective proliferation (Podolska et al., 2012; Sakuma et al., 2012). In the current study, an effective siRNA target sequence to hIRF7 was confirmed and a derivative lentiviral delivering vector containing this siRNA-expressing cassette was developed to express the target sequence in the appropriate cells or tissues. Upon insertion of the siRNA transcription cassette into the vector, the siRNA can be produced. Based on this shuttle plasmid and helper lentivirus packaging plasmid, a recombinant RNA interference lentivirus was prepared. After infecting cells, siRNAs for the target gene were stably expressed and were functional for a long period. Using the screening plasmid psiSDR, we successfully identified the strongest-interfering siRNA (hIRF7-i1) for the target gene hIRF7 from among three siRNA transcription templates. We then constructed hIRF7-i1-carrying recombinant lentivirus (LV-siRNA-hIRF7) to target hIRF7 using the modified lentivirus shuttle plasmid pLenti7.3/V5TOPO. Subsequent functional analysis showed that LV-siRNA-hIRF7 effectively silenced hIRF7 gene expression through RNA interference, and inhibited the differentiation of monocytes to macrophages. To validate our system that can work well, we also screened and indentified two optimal siRNAs for human TLR4 gene. In summary, we have developed a dual luciferase assay-based siRNA screening method and demonstrated its utility for evaluating the geneknockdown efficiency of candidate siRNA sites (Fig. 5). The luciferasebased assay for gene-silencing efficiency is both qualitative and quantitative. Reduction of relative luciferase activity is closely correlated to knockdown efficiency on the expression and functional activity of target genes. Additionally, the derivative lentivirus delivery system provides a highly efficient means to transduce optimal siRNA to target cells. Thus, the psiSDR and derivative lentivirus delivery system is an efficient, versatile, yet user-friendly tool for selecting, validating, and delivering optimal siRNA sites for RNA-mediated gene silencing.

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Fig. 5. Schematic overview of the user-customizable procedure for rapid screening of effective siRNA target sites by one-step transfection with a single double-cloned psiSDR vector, and the development of a derivative lentivirus delivery system for further assessing appropriate biological effects after target gene knockdown.

Acknowledgments The authors thank Dr. Luo Qing, Pediatrics Institution of Chongqing Medical University, Chongqing, China for providing the pSOS-HUS vector. This work was supported by the National Natural Science Foundation of China (Nos. 81000834; 31070792) and the China Postdoctoral Science Foundation funded project (20070420769). Requests for psiSDR plus related vectors and their full sequences should be directed to [email protected].

References Conflicts of interest The authors declare no conflicts of interest.

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