Acta Scientiae Veterinariae ISSN: 1678-0345 [email protected] Universidade Federal do Rio Grande do Sul Brasil

Simiano Tavares, Kaio César; de Mello e Pinho, Raquel; de Sá Carneiro, Igor; de Aguiar, Luís Henrique; Méndez Calderón, Carlos Enrique; Tondello Martins, Leonardo; Ambrósio, Carlos Eduardo; Maga, Elizabeth Anne; Bertolini, Marcelo; Murray, James Donald; Relly Bertolini, Luciana Efficient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA) Acta Scientiae Veterinariae, vol. 40, núm. 3, 2012, pp. 1-11 Universidade Federal do Rio Grande do Sul Porto Alegre, Brasil

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ORIGINAL ARTICLE ISSN 1679-9216 (Online) Pub. 1048

Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA)

Kaio César Simiano Tavares1, Raquel de Mello e Pinho1,2, Igor de Sá Carneiro1, Luís Henrique de Aguiar1, Carlos Enrique Méndez Calderón1, Leonardo Tondello Martins1, Carlos Eduardo Ambrósio2, Elizabeth Anne Maga3, Marcelo Bertolini1, James Donald Murray3 & Luciana Relly Bertolini1

ABSTRACT

Background: RNA interference (RNAi) is a post-transcriptional silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant pro- teins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) were chosen to generate dsRNA for subsequent “Dicing” to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80oC. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specifi c gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confl uence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post- for protein determination. The knockdown of specifi c targeted gene products was quantifi ed by Western blot using GAPDH as control. Transfection of gene-specifi c siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for or knockout studies. Stealth RNAiTM was developed to achieve high specifi city and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Keywords: RNA interference, post-transcriptional gene silencing, Stealth RNAiTM, non-homologous end joining.

Received: May 2012 www.ufrgs.br/actavet Accepted: June 2012 1Molecular and Lab, School of Medicine, University of Fortaleza, Fortaleza, CE, Brazil. 2Molecular and Developmental Mor- phophysiology Lab, School of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil. 3Transgenics Lab, Department of Animal Science, University of California, Davis, CA, USA. CORRESPONDENCE: L.R. Bertolini [[email protected] - Tel.: +55 (85) 9628-6893]. Molecular and Developmental Biology Lab, Health Sciences Center, University of Fortaleza (UNIFOR). Av. Washington Soares n. 1321, Bairro Edson Queiroz. CEP 60.811-905 Fortaleza, CE, Brazil.

1 K.C.S. Tavares, R.M. Pinho, I.S. Carneiro, et al . 2012. Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA). Acta Scientiae Veterinariae. 40(3): 1048.

INTRODUCTION MATERIALS AND METHODS

RNA interference (RNAi) is a naturally occur- In vitro production of siRNA fragments (Diced siRNA) ring post-transcriptional gene silencing mechanism that was fi rst described in [14], but The system for the preparation of in vitro- subsequently shown as a very conserved process in a transcribed siRNA fragments, along with the strategy wide range of cell types and [9,40,47]. Small for the use of the RNAi cell machinery are illustrated interfering double stranded RNA fragments (siRNAs) in Figure 1. In this study, two sets of siRNA mixtures for each gene (Ku70 and Xrcc4) were prepared, as are the cell mediators of the RNAi mechanism, and as follows. Fragments of siRNA were in vitro-produced such, siRNAs can be used in in vitro and in vivo studies using primers to amplify DNA fragments from two for RNAi-induced specifi c post-transcriptional gene different sequence regions for the Ku70 and Xrcc4 hu- silencing [12,14,22,40,44]. man genes (Table 1). Briefl y, genomic DNA (gDNA) Fragments of siRNA tailored for gene- purifi ed from HCT116 cells1 were subjected to 30 specifi c silencing can be synthesized in a number PCR cycles, as follows: 95°C for 30 s, 56°C for 30 of ways, including the chemical synthesis, the in s, 72°C for 45 s, and one cycle at 72°C for 7 min. vitro or enzymatic synthesis by T7 phage RNA poly- After PCR amplifi cation, amplicons were cloned merase from DNA templates, and the into the pCRTMII-TOPO® cloning vector2. DNA was in vivo or vector-based synthesis within living cells purifi ed using the Wizard® plasmid purifi cation kit3 [1,5,19,22,23,49]. Several methods based on the use and sequenced at a commercial facility4. Searches of of siRNA mixtures have been developed to increase the human database were carried out to ensure that targeting chances by siRNAs, including the prepara- the chosen sequences would not target other gene tion of siRNA mixtures using either RNase III [48] or transcripts. Double-stranded RNAs (dsRNAs) were Dicer enzymes to digest longer double-stranded RNAs produced using the BLOCK-iTTM T7-TOPO® Linker2, [3,26]. The siRNA mixtures from such digestions according to the manufacturer’s instructions. In brief, have been found highly effi cient at inducing RNAi the T7 promoter2 was used to generate sense and anti- knockdown effects in living cells [3,11,18,39,41]. sense DNA templates from the amplicons obtained for As the RNAi effectiveness of distinct siRNA each gene of interest. In vitro transcription reactions types may differ signifi cantly among preparations, were done for each strand using the RNA Transcrip- the aim of this study was to compare the knockdown tion Kit2. Single-stranded RNAs (ssRNAs) were puri- effects of in vitro-produced (Diced) and chemically fi ed with the BLOCK-iTTM RNA Purifi cation Kit2, in synthesized (Stealth RNAiTM) siRNAs on the rela- vitro-annealed to generate long dsRNAs, and diced tive abundance of Ku70 and Xrcc4 gene products in using the BLOCK-iTTM Dicer RNAi Kit2 to produce human cells. Both proteins were chosen for their short fragments of double-stranded small interfering high levels in cells and their key roles in the non- RNAs (ds-siRNAs or siRNAs). Diced siRNAs for homologous end joining (NHEJ) DNA repair pathway both genes were quantifi ed and stored at –80oC. The by binding and stabilizing DNA double strand breaks siRNA fragments produced for the two gene sequence (DSBs) and mediating the DNA end joining and liga- regions for each gene were pooled in equal amounts, tion processes [8,13,20,27,28,45,46]. by gene, prior to use in cell transfection.

Table 1. Target gene sequences, primers and GenBank accession numbers used for the in vitro production of siRNA fragments (Diced siRNA).

Position in GenBank siRNA Target sequence Sense strand (5’-3’) Antisense strand (5’-3’) the gene accession no.

Ku70-1 167 bp, exon 5 ACGAATTCTAGAGCTTGACCAGTT TGAACAGCATGATCCTCTTATGAC 407 to 574 NM001469 Ku70-2 249 bp, exon 13 TATTCAGAAGAGGAGCTGAAGACC CCTAGCTCAGGAGAAACACATTTT 1764 to 1989

Xrcc4-1 172 bp, exon 2 CTGAATCAGAGATTTCCCAAGAAG GAGACATCTTTCAGGTTTTTCTCA 317 to 489 NM003401 Xrcc4-2 108 bp, exon 6 GATGTCACTGATATTGCACCAAGT TTCCTTTTCTTGAAGCTGATTCTC 858 to 1067

2 K.C.S. Tavares, R.M. Pinho, I.S. Carneiro, et al . 2012. Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA). Acta Scientiae Veterinariae. 40(3): 1048.

Figure 1. Major steps for the in vitro production of siRNA fragments (Diced siRNA) and for the strategic use of the RNAi pathway machinery in the cell. RNAi is a post-transcription gene silencing mechanism through the degradation of homologous messenger RNA (mRNA) induced by short interfering RNA (siRNA). Once in vitro-transcribed, percursor long double stranded RNA (dsRNA) is recognized and processed by the enzyme Dicer. The mixture of double-stranded processed siRNAs is transfected into cells where the RNA-induced silencing complex (RISC) binds and unwinds the siRNA, ultimately binding to homologous target mRNA for degradation, resulting in protein depletion. Modifi ed from Bertolini L.R. & Bertolini M. [2].

Chemically Synthesized siRNA fragments (Stealth RNAiTM) concentration (data not shown) was selected for experi- Two specifi c siRNA (Stealth RNAiTM 2) frag- mental use, as below. ments for two distinct regions of each targeting gene Gel electrophoresis for DNA and siRNA analyses (human Ku70 and human Xrcc4; same GenBank acces- Amplicons were separated in 1.5% agarose gels sion numbers as in Table 1), were designed using the containing ethidium bromide, in TBE buffer. Stealth Invitrogen algorithm system5 and commercially synthe- siRNAs and Diced siRNAs for Ku70 and Xrcc4 were sized2. RNA sequences for Ku70 siRNA Stealth RNAiTM analyzed on 2% and 4% agarose gels, respectively. A fragments (Ku70 Stealth siRNA) were: Ku70-1 sense 100 bp DNA step ladder2 and a 10 bp DNA ladder2 were 5’-CCUCCAAUAAAGCUCUAUCGGGAAA-3’; and used in the experiments as molecular weight markers. Ku70-2 sense 5’-GGAGUCGUCAGAUUAUACUG- GAGAA-3’. RNA sequences for Xrcc4 siRNA Stealth Cell cultures and cell transfections with Stealth or RNAiTM fragments (Xrcc4 Stealth siRNA) were: Xrcc4-1 Diced siRNAs sense 5’-CCACCUUGUUUCUGAACCCAGUAUA-3’; Human (male) colon cancer HCT116 cells1 and Xrcc4-2 sense 5’-GGAAGCUUUGGAGA- were grown at 37°C and 5% CO2, in McCoy 5A me- CUGAUCUUUAU-3’. A nonspecifi c Stealth siRNA2 dium6 supplemented with 10% fetal calf serum (FCS)7, non homologous to any known gene (5´-UAAAU- and 1% antibiotic-antimycotic solution containing GUACUGCGCGUGGAGAGGAA-3´) was used as a 10.000 IU sodium penicillin, 10 mg streptomycin control. Also, a BLOCK-iTTM fl uorescein-conjugated sulfate and 25 µg amphotericin B7. Cells were seeded siRNA2 was used to verify transfection effi ciency. The in six-well plates at 40-50% confl uence 24 h prior two Stealth siRNAs for each gene product (Ku70 and to siRNA transfection. Cells were treated according Xrcc4) were tested after cell transfection, and only the to the following siRNA treatments: (1) 0, 250, 500, siRNA for each gene that achieved the most effi cient 1000 and 1500 nM Diced (pooled) siRNAs for Ku70 protein knockdown with the smallest or Xrcc4 gene products; (2) 40 nM (Ku70) and 20

3 K.C.S. Tavares, R.M. Pinho, I.S. Carneiro, et al . 2012. Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA). Acta Scientiae Veterinariae. 40(3): 1048. nM (Xrcc4) Stealth siRNAs for Ku70 and Xrcc4 gene radish peroxidase (1:1000) for 1 h at RT. Membranes products; (3) 100 nM nonspecifi c Stealth siRNA (non- were washed three times in TBS-T, two times in TBS, specifi c siRNA control); (4) blank or sham transfection and developed using the ECL detection kit9. Images (no siRNAs; transfection control); and (5) 100 nM were recorded and band intensities on western blots fl uorescein-conjugated siRNA (marker for transfec- were quantifi ed using the CCD-based FluorChemTM tion effi ciency). Cell transfections with siRNA frag- 8000 Imaging System and Alphaease FC Software13. ments were performed in triplicates, for each siRNA treatment and concentration as above, using 2 µg/mL Flow cytometry assay for GFP-specifi c Stealth siRNA activity in GFP-expressing cells Lipofectamine 20002, according to the manufacturer’s instructions. Lipid-RNA complexes were prepared in To determine the effectiveness and repeatability OptiMEM I2 according to the manufacturer’s instruc- of the chemically synthesized method for RNAi-induced tions to be added in a drop-wise manner to the cells protein down regulation, including siRNA doses and (500 µL/well) in 6-well plates, followed by a 4 to 6 h time period for the phenotypic effects on cells, a set o of experiments was carried out with the use of distinct incubation at 38.5 C and 5% CO2. Finally, cells were overlaid with 2 mL culture medium containing 15% Stealth siRNA sequences to attenuate specifi cally the FCS (12% fi nal volume). At least three independent expression of a reporter gene (green fl uorescent protein, transfection experiments (replications) were performed GFP) in cells in culture. A GFP expression vector driven for each siRNA treatment and concentrations. by the CMV promoter (pEGFP-N1)14 was linearized Cells transfected with fluorescein-labeled with Afl II2, resulting in a 4.8 kb DNA fragment. The siRNA, used as a control for transfection effi ciency, linearized plasmid (0.5 µg per well for 24-well plate) were analyzed 5 h post-lipofection by fl uorescence was transfected into HCT116 cells (control cells) or microscopy using a fl uorescein isothiocyanate fi lter co-transfected with 40 nM of six distinct chemically set. The effects of Ku70 and/or Xrcc4 Diced or Stealth synthesized GFP-specifi c Stealth siRNA sequences siRNA treatments and the sham and nonspecifi c siRNA (GFP Stealth siRNA), using the same procedures as controls on protein abundance were determined by for Ku70 and Xrcc4 Stealth siRNAs. Trypsinized and Western blot at 24, 48, 72 and 96 h after transfection. paraformaldehyde-fi xed (1%, w/v) GFP-expressing cells were analyzed by fl uorescence-activated cell sorting Western blot analysis using a FACScan machine15 with Cell Quest software HCT116 cells treated with different doses of 48 h after linear pEGFP-N1 and/or GFP Stealth siRNA Ku70, Xrcc4 or nonspecifi c siRNAs, as well as cells transfections. The proportion of GFP-positive cells was 7 from sham transfections, were rinsed with PBS and scored in dot-plots of GFP fl uorescence versus FL3 lysed with 200 µL radioimmunoprecipitation (RIPA) autofl uorescence, using mock-transfected (sham) cells buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% to defi ne the negative area. A minimum of three inde- NP-40, 0.25% sodium deoxycholate). Protein concen- pendent experiments conducted in duplicate for each trations in each cell sample were measured using the GFP Stealth siRNA treatment were preformed. Bradford Protein Assay8. Equal amounts of protein (5 µg) from each sample were mixed with 2x SDS Data analyses buffer8, heated at 95°C for 5 min, and fractionated by Protein amounts obtained by densitometry 10% SDS–PAGE. Gels were then transferred to Hy- were normalized relative to the amount of GAPDH bond P nylon membranes9, blocked with TBS-T (25 in the sample. Flow cytometry data were normalized mM Tris-HCl pH 7.6, 25 mM NaCl, 0.25% Tween-20) to control GFP cell values. Comparisons were made16 containing 5% nonfat dry milk, and probed with by constructing 95% confi dence intervals on data primary monoclonal antibody to Ku70 (MS-329)10; regarding protein analysis by densitometry and GFP polyclonal anti-Xrcc4 (sc-8285)11; and a monoclonal expression by fl ow cytometry, for P < 0.05. anti-GAPDH antibody (ab9484)12 as a loading control RESULTS in TBS-T containing 1% nonfat dry milk for 2 h at RT. After washing three times in TBS-T, membranes were All Diced siRNAs produced in this study further incubated with the appropriate anti-rabbit or had multiple 21-23 bp double-stranded RNA frag- anti-mouse secondary antibodies11 conjugated to horse- ments corresponding to each one of the targeted 4 K.C.S. Tavares, R.M. Pinho, I.S. Carneiro, et al . 2012. Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA). Acta Scientiae Veterinariae. 40(3): 1048. gene sequences, for each gene of interest (Figure 2). and 3c). Since the latter level of inhibition was com- Agarose gel electrophoresis (Figure 2a) confi rmed parable to the overall cell transfection efficiency the correct size of resulting amplicons for each gene (~90%), it seems that Ku70 expression was almost sequence of interest, which were cloned into the completely inhibited in the population of success- pCRTMII-TOPO® cloning vector and also sequenced fully transfected cells at 500 nM concentration and to ensure precise templates for transcription. Two at 48 h post-transfection (Figure 3a). Similarly, a different DNA fragments for each gene (Table 1) 250 nM Xrcc4 siRNA dose caused a knockdown generated pools of distinct siRNA-coding sequences; of approximately 80% in the Xrcc4 protein level in each siRNA-coding 21-23 mer fragments containing cells (48 h; Figure 3b and 3d). As expected, greater 100% correct sequences. concentrations of Dicer Ku70 or Xrcc4 siRNAs After purifi cation (Figure 2d), the typical fi nal did not further reduce the Ku70 or Xrcc4 protein concentration of the Diced siRNA mixtures obtained abundances in transfected cells (Figure 3). from a single preparation were 20-45 µM (or 30-60 µg As the greatest knockdown effect was ob- total siRNA in 300 µL), which was deemed suffi cient served at 48 h post-transfection, a 500 nM Dicer for many cell culture transfection experiments. The Ku70 and a 250 nM Xrcc4 siRNA doses were cho- electrophoretic mobility of the Diced siRNA mixtures sen as standard concentrations for subsequent cell was positioned between the 20 and 25 bp DNA mark- transfection experiments. When cells were trans- ers, which corresponded to the expected 21-23 mer fected with 500 nM Ku70 siRNA and 250 nM Xrcc4 siRNA mobility, with two overhanging nucleotides at siRNA, Ku70 and Xrcc4 expression levels started to each 3’ RNA end (Figure 2d). decline at 24 h, with the highest decrease observed As observed after the fluorescent-labeled at 48 h, with the return to control levels by 96 h after siRNA and GFP cell transfections, based on the detec- siRNA transfection (Figure 3b). The non-specific tion of the fl uorescent labeling 4 h after transfection, siRNA-treated cells had no effect on the Ku70 and and 24 h after transfection, respectively, transfection Xrcc4 protein levels.The same successful level of effi ciency was similar in all cell samples, with more down-regulation obtained with Dicer siRNAs was than 90% of the cells taking up fl uorescent siRNA also observed with the use of Stealth siRNAs at 48 fragments after transfection. h post-transfection. However, the amount required The functional activities of different doses of to achieve a 90% knockdown effect was 40 nM Diced siRNAs (Figure 3), prepared using DNA frag- for Ku70 and 20 nM for Xrcc4, which was 10-fold ments (Table 1), and of Stealth siRNAs (Figure 4) for lower than with in vitro-produced Dicer siRNA Ku70 and Xrcc4 protein depletion were examined on fragments. Also, Ku70 and Xrcc4 protein abun- cells every 24 h for up to 96 h post-transfection. Cells dances in Stealth siRNA-trated cells were normal transfected with no siRNA (sham, lipofectamine only), nonspecifi c siRNA, and Diced or Stealth Ku70 siRNAs by 96 h after Stealth siRNA transfection (Figure and/or Xrcc4 siRNAs at different concentrations were 4). It is evident from western blot control data and tested by western blotting for the amount of attained from a visual inspection of treated cells that Diced protein knockdown, after normalization to GAPDH siRNA mixtures did not show cytotoxic effects at protein abundance level. the concentrations used in this study. The effect of Diced Ku70 and Xrcc4 The fl ow cytometry assay for GFP-specifi c siRNAs on Ku70 and Xrcc4 protein knockdown, Stealth siRNA activity revealed that GFP expression respectively, followed a dose-dependent man- in all groups of GFP-specifi c siRNA-treated cells was ner, with no reciprocal attenuation effect of one signifi cantly different from control cells and non- siRNA system on the other. When compared specifi c siRNA-treated cells at 48 h post-transfection with the sham and non-specific controls 48 h (Figure 5). Two siRNA sequences were effective in after siRNA transfection, a 250 nM Ku70 siRNA down regulating approximately 80% of the expression dose reduced endogenous Ku70 protein by 70%, in GFP-expressing HCT116 cells, with close to 90% whereas Ku70 expression was inhibited by 80 to attenuation levels observed only for two GFP Stealth 90% at the 500 nM Ku70 siRNA dose (Figure 3a siRNA sequences (P < 0.05).

5 K.C.S. Tavares, R.M. Pinho, I.S. Carneiro, et al . 2012. Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA). Acta Scientiae Veterinariae. 40(3): 1048.

Figure 2. Diced siRNA preparation and purifi cation. (a) Amplicons for Ku70 (lanes 1 and 2) and Xrcc4 (lanes 3 and 4). (b) In vitro-produced siRNA fragments representing 200 and 300 bp regions of the Ku70 gene, and 150 and 100 bp regions of the Xrcc4 gene. The sense and antisense strand for each region were annealed, showing a band biger in size than the amplicons. A high molecular weight smear is visible due to branched annealing products. Lanes - M: 100 bp DNA ladder, with reference values, in bp, on the left; 1: Ku70-1 sense transcript, 2: Ku70-1 antisense transcript, 3: Annealed Ku70 dsRNA; 4: Xrcc4-1 sense transcript; 5: Xrcc4 antisense transcript, 6: Annealed Xrcc4-1 dsRNA. (c) Enzymatic digestion (Diced) Ku70 and Xrcc4 ds-siRNA products prior to purifi cation. Lanes - M: 10 bp DNA ladder. (d) Purifi ed siRNA products ready for use. Lanes - M: 10 bp DNA ladder, with reference values, in bp, on the left.

Figure 3. Endogenous Ku70 or Xrcc4 protein abundances in HCT116 cells after transfection with Diced siRNA fragments, as quantifi ed by western blotting. (a) Cells transfected with different Diced Ku70 siRNA concentrations and harvested 48 h after transfection. (b) Cells were transfected with 500 nM Diced Ku70 siRNAs (top blot) and 250 nM Diced Xrcc4 siRNAs (bottom blot) and harvested 48 and 96 h after trans- fection. (c) and (d) Normalized values for Ku70 and Xrcc4 protein relative abundances, relative to non-transfected control cells, at 48 and 96 h post-transfection with Dicer siRNA. Normalized values for Ku70 or Xrcc4 protein relative abundances were relative to non-transfected control cells. C: Non-transfected control cells; NS: non-specifi c siRNA-transfected cells.

Figure 4. Stealth siRNA-mediated down-regulation of Ku70 and Xrcc4 proteins in HCT116 cells, transfected with 40 nM Stealth Ku70 siRNA and 20 nM Stealth Xrcc4 siRNA, and harvested at 24, 48, 72 and 96 h post-transfection. (a) Ku70 and (b) Xrcc4 Western blots from cells col- lected 48 h after Stealth siRNA transfection. (c) Ku70 and (d) Xrcc4 protein levels up to 96 h post-transfection, expressed as percentage values, after normalization against GAPDH. C: Non-transfected control cells; NS: non-specifi c siRNA-transfected cells.

6 K.C.S. Tavares, R.M. Pinho, I.S. Carneiro, et al . 2012. Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA). Acta Scientiae Veterinariae. 40(3): 1048.

Figure 5. GFP-expressing HCT116 cells (%), relative to controls (GFP cells), determined by fl ow cytometry 48 h following co-transfection of a linear GFP expression vector and six distinct GFP-specifi c Stealth siRNA sequences. GFP cells: Non-treated GFP control cells; NS-siRNA: Non-specifi c siRNA-transfected GFP cells; Stealth 1 to 6: transfected GFP cells with six distinct GFP-specifi c Stealth siRNA sequences (1 to 6). a,b,c,d:Columns without common letters differ (P < 0.05).

DISCUSSION ‘background’ genome) could be excluded from a set of RNA interference has emerged as an alternative siRNAs for optimal gene-specifi c targeting [16,32,36]. The discovery that RNAi is effective and and rather effi cient method for functional genomics can be used to transiently silence in studies, having the potential to become an important mammalian cells has opened new avenues to study tool to enhance livestock production through the proteins in mammalian cells for genes for which increase in production effi ciency, the prevention of knockout models are non-viable [6,10,43] such as is the diseases, for the development of therapeutic drugs, case for DNA repair proteins. The use of RNAi allows particularly anti-viral therapies, and also to enhance the further investigation of DNA repair proteins in production of transgenic animals [2]. Proper design cancer research and also exogenous DNA integration. and optimal transfection conditions are critical for The present study demonstrated a successful transient high levels of gene knockdown in RNAi experiments depletion of Ku70 and Xrcc4 proteins, obtained by [3]. Results presented in this study demonstrated that the transfection of siRNA fragments into HCT116 the chosen target sequences and both siRNA prepa- cells. DNA repair proteins play a major role in the ration methods were effi cient for the production of response to DNA damaging agents in mammalian cells functionally active siRNA fragments. To conduct [17,25] making them attractive targets considering a proof-of-principle experiment for effective gene the importance of DNA repair enzymes for DNA knockdown using two distinct RNAi approaches, we damage control. Our results showed that a pool of targeted Ku70 and Xrcc4, two highly expressed genes enzymatically produced Ku70 or Xrcc4 Diced siRNAs in mammalian cells known to regulate DNA damage could be as effectively used to obtain a transient and DNA integration in transgenic experiments [4,29]. depletion of these essential proteins as Stealth siRNA, We also successfully targeted GFP expression in GFP- without any detectable detrimental or side effects on the expressing cells with GFP Stealth siRNAs, confi rming cells, with the expectation that such a depletion by both the repeatability and the robustness of the chemically approaches would lead to loss-of-function phenotypes. synthesized Stealth siRNAs. In our design of the Ku70 The testing of various concentrations on and Xrcc4 siRNA fragments, we chose only gene frag- siRNAs allowed the determination of optimum siRNA ments that contained no homology with other published doses (400 nM for Ku70 and 200 nM for Xrcc4 for human gene sequences, thereby expecting to minimize Diced siRNAs; 40 nM for Ku70 and 20 nM for Xrcc4 non-specifi c effects on cells. Hence, any potentially for Stealth siRNAs) that yielded the maximum protein cross-reactive sequences (e.g., sequences that contain knockdown effects. Furthermore, the time course homology to closely related known targets or, per- analysis showed that maximum depletion of the haps, those having minimal homology to a particular targeted protein was attained by 48 h post-transfection,

7 K.C.S. Tavares, R.M. Pinho, I.S. Carneiro, et al . 2012. Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA). Acta Scientiae Veterinariae. 40(3): 1048. with protein levels returning to near normal values by In summary, the present study demonstrated 96 h post-transfection. These fi ndings are consistent the successful transient depletion of the human with previous data reporting the half-lives of these Ku70 and Xrcc4 proteins using two distinct RNAi proteins to be in the range of 16 to 24 h [15,33]. approaches. The Diced or enzymatically produced The Diced siRNA mixtures significantly siRNA was the most cost effective and quickest reduced Ku70 and Xrcc4 protein levels, but Western method, being mediated by the T7 phage RNA blot analyses showed that cellular Ku70 or Xrcc4 polymerase in vitro transcription from short DNA expression could not be completely depleted. In fragments [38], whereas the chemically synthe- fact, fragments of siRNA act on cells by specifically sized stealth siRNA represented the gold standard inducing degradation of the mRNA for the targeted for RNAi. Chemically synthesized siRNAs can be gene, being unlikely to cause a complete transcript produced with a wide range of chemical modifi ca- and protein depletion, with the attaining of a knock- tions, making them more stable, more specifi c and down or attenuating effect [12]. Transfection with consistent, as observed in this study. The down side, 250 nM Ku70 siRNAs down regulated around 70% however, of its use include the higher manufacturing of the Ku70 protein levels seen in the respective costs and period of time required for the synthesis mock-transfected cells. However, transfection with of Stealth siRNA fragments. increased concentrations of Ku70 siRNA (500 nM) CONCLUSIONS reduced the protein levels to 10% of the amount seen in the mock-transfected cells. Notably, in- Results from this study demonstrated the ef- creasing the concentration of siRNA even further fectiveness of enzymatically produced (Diced) siRNAs did not cause a complete depletion of the protein, in inhibiting abundant target proteins in cultured cells agreeing with observations by Elbashir et al. in a manner similar to that induced by chemically [12]. Each efficient siRNA has an optimum dose- synthesized (Stealth) siRNAs. The in vitro production response curve relative to a maximal knockdown of siRNA mixtures is more cost effective when com- effect, which was, in this study, in the range of 500 pared with the chemical synthesis in the production nM for Ku70 and 250 nM for Xrcc4. Altogether, of Stealth siRNAs. As mixtures may contain a wide range of distinct Diced siRNA sequences, effective the transient depletion of essential cell proteins doses to attain high protein knockdown levels (90%) over a time period of several days, without observ- were 10-fold higher than with the use of target-specifi c able negative effects on cell survival, suggests the Stealth siRNA fragments. Also, the in vitro method possibility of carrying out a series of sequential provided siRNA fragments in larger scale, suitable for transfections to elucidate the function of genes or pharmaceutical applications or for the production of manipulate cellular machinery for therapeutic or RNA libraries, which would be complex to be attained other end uses [7,31,42,50]. with chemically synthesized Stealth siRNA fragments. Like other classes of antisense agents for se- quence-specifi c mRNA knockdowns, such as antisense SOURCES AND MANUFACTURES 1 or ribozymes, effective application of American Type Culture Collection (ATCC), Manassas, VA, USA. 2Invitrogen Corporation, Carlsbad, CA, USA. RNAi in mammalian cells requires effi cient delivery, 3Promega, Madison, WI, USA. identifi cation of sensitive sites in the target RNA se- 4Davis Sequencing Facility, Davis, CA, USA. 5 quence, and minimization of off-target effect [30,35]. www.invitrogen.com/rnaidesigner 6Sigma-Aldrich, Saint Louis, MO, USA. Two types of off-target effects have been described in the 7Gibco-BRL®, Grand Island, NY, USA. literature: (i) a non-specifi c effect, which is independent 8Bio-Rad, Hercules, CA, USA. of sequence homology with a particular siRNA, and (ii) 9GE Healthcare Biosciences, Pittsburg, PA, USA. 10Neomarkers, Fremont, CA, USA. a specifi c effect, when non-targeted genes with partial 11Santa Cruz Biotechnology, Santa Cruz, CA, USA. complementarity to a particular siRNA directed against 12Abcam, Cambridge, MA, USA. the target gene are down-regulated [21,24,34,37]. In the 13AlphaInnotech Corp., Santa Clara, CA, USA. 14Clontech, Mountain View, CA, USA. present study, we did not investigate whether our siRNA 15Becton Dickinson, Franklin Lakes, NJ, USA. mixtures would cause potential off-target effects. 16Minitab Statistical Software, State College, PA, USA.

8 K.C.S. Tavares, R.M. Pinho, I.S. Carneiro, et al . 2012. Effi cient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA). Acta Scientiae Veterinariae. 40(3): 1048.

Acknowledgments. The authors thank Dr. Knut R. Madden for Declaration of interest. The authors report no confl icts of advice on the experiments and for supplying the Stealth siRNAs. interest. The authors alone are responsible for the content and This study was supported by the UC Discovery Grant Bio03- writing of the paper. 10412, in partnership with Invitrogen Corporation.

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