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APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Development of siRNA expression vector utilizing rock bream β-actin promoter: a potential therapeutic tool against viral infection in fish
Kosuke Zenke & Yoon Kwon Nam & Ki Hong Kim
Received: 30 June 2009 / Revised: 27 July 2009 / Accepted: 30 July 2009 / Published online: 15 August 2009 # Springer-Verlag 2009
Abstract In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream β-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream β-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream β-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species. Keywords RNA interference . β-actin promoter . Short interfering RNA . Fish cell . Rock bream iridovirus
Introduction RNA interference (RNAi) is a cellular mechanism induced by double-stranded RNA for posttranscriptional gene silencing (Hannon 2002). In mammalian cells, short interfering RNAs (siRNAs), an inducer for RNAi, can be expressed from plasmids or viral vectors as a short hairpin RNA (shRNA). Although shRNA can be generally transcribed using RNA polymerase III promoter such as U6 small nuclear RNA promoter (U6 promoter), several studies have shown that simple modification of RNA pol. II promoter such as early immediate cytomegalovirus promoter (CMV promoter) also enabled the expression of effective siRNA. Xia et al. (2002) reported that siRNA was successfully expressed in mammalian cells using modified CMV promoter which is a typical RNA pol. II promoter widely used for exogenous gene expression system of eukaryotes. In fish species, the same construct using CMV promoter was reported to be used to effectively express siRNA and silence the gene expression in zebra fish embryos (Du et al. 2006; Su et al. 2008a, b). The β-actin is one of the most abundant proteins ubiquitously expressed in all types of eukaryotic cells, and β-actin promoter of mammalians and birds has been precisely characterized (Kawamoto et al. 1988; Quitschke et al. 1989; Danilition et al. 1991) and applied to express exogenous genes (Miyazaki et al. 1989; Williams et al. 1991; Zhang et al. 1994). In fish species, β-actin promoter was first described in common carp (Cyprinus carpio; Liu et al. 1990a) and shown to have activity to express exogenous genes (Liu et al. 1990b). While isolation and potential applicability of piscine β-actin promoter have been reported in zebra fish (Danio rerio; Higashijima et al. 1997), tilapia (Oreochromis niloticus; Hwang et al. 2003), and two cyprinid species (Noh et al. 2003; Feng et al.
K. Zenke : K. H. Kim (*) Department of Aquatic Life Medicine, Pukyong National University, Nam-gu 599-1, Busan 608-737, South Korea e-mail: firstname.lastname@example.org Y. K. Nam Department of Aquaculture, Pukyong National University, Busan 608-737, South Korea
Orientation of CArG insert was confirmed by sequencing and named as pβAcoreCArG(+)-EGFP (3. one recent study has shown that transcription activity of carp β-actin promoter in epithelial papulosum cyprini (EPC) cells was about two times stronger than that of CMV promoter (Ruiz et al. Orientation of CArG insert (opposite direction) was confirmed by sequencing and named as pmβA. which was named pβAcore-SV40mpA. Finally. modified CMV promoter-driven EGFP expression vector was constructed by ligating the EGFP gene sequence fragment PCR-amplified with EGFP F2 and EGFP R2 from pEGFP-C1 to pSilencer4. opposite direction). For comparison purposes. pβA-EGFP (1) driven by fulllength β-actin promoter was described in the previous study (Zenke and Kim 2008). the 278-bp fragment of enhancer region containing CArG motif was excised from pβA-EGFP by NcoI digestion and ligated to NcoI-digested pβAcore-SV40mpA. 1 M NaCl. Finally. which enabled the successful siRNA expression from plasmid in fish cell lines (Zenke and Kim 2008). For this. and several clones were sequenced at both directions using ABI 3700 (PE Applied Biosystems). siRNA expression vector utilizing the modified rock bream β-actin promoter was constructed. Annealed oligonucleotide was then ligated to pmβA digested with . 2a. First. 10 mM EDTA) and allowed to cool down at room temperature for 30 min. possible use of the newly developed siRNA expression vector for inhibition of virus replication was examined. For this. Materials and methods Sequence analysis of rock bream β-actin promoter All oligonucleotides used in the present study are summarized in Table 1. respectively. II promoter of fish species for expression of siRNA in fish cells and to develop another effective vector system. After removal of vector and primer sequences. which was named pβAcore-EGFP (2). FJ975146 and FJ975145. we have reported the development of fugu U6 promoter-driven siRNA expression vector.5).1 (Ambion) using SV40mpA F (including HindIII and BglII site) and SV40mpA R (including SpeI site) and ligated to downstream of core promoter using BglII site and SpeI site contained in pGEM T easy vector. Construction of EGFP expression vector driven by modified β-actin promoter EGFP expression vectors constructed in the present study were schematized in Fig. the longest cDNA sequence obtained was aligned with rock bream β-actin gene sequence. RACE polymerase chain reaction (PCR) products were subcloned into pGEM T easy vector (Promega). and knockdown efficiency of a reporter gene enhanced green fluorescent protein (EGFP) was compared with siRNA expression vector utilizing modified fugu U6 promoter in grunt fin cells (GF cells). the fragment containing the part of core promoter and EGFP gene sequence was PCR-amplified by two-step PCR from pEGFP-C1 (Clontech) using EGFP R and two overlapping forward primer pBAcore-EGFP F1 and F2 which contained EGFP gene and the part of core promoter. Construction of siRNA expression vector targeting EGFP siRNA target sequence for EGFP (5′-AAGCTGACCCT GAAGTTCATC-3′. In order to examine the use of RNA pol. the 278-bp fragment of enhancer region containing CArG motif was excised from pβA-EGFP by digestion with NcoI and ligated to pβAcore-EGFP digested with the same restriction enzyme. which was named pβAcoreCArG(-)d-EGFP (5). we constructed siRNA expression vector utilizing pβAcore-SV40mpA. Next. EGFP gene sequence including kozak sequence and SV40 polyadenylation signal was excised from pβA-EGFP with HindIII and SpeI digestion and ligated to pβAcoreSV40mpA digested with the same restriction enzyme. respectively). One microgram of purified rock bream spleen RNA was then subjected to RACE reactions using SMART cDNA RACE kit (Clontech) according to manufacturer ’s instruction. which was named pCMVm-EGFP (6). For the determination of transcription start site (TSS) of rock bream β-actin promoter. Full-length β-actin gene and cDNA sequence of rock bream were obtained from GenBank (GenBank accession nos. Core promoter region of rock bream β-actin promoter was PCR-amplified using pβAcore F and pβAcore R and subcloned into pGEM T easy vector. 2008). SV40 minimal polyadenylation signal (SV40mpA) sequence was also PCRamplified from pSilencer 4.680 Appl Microbiol Biotechnol (2010) 85:679–690 2006). we have characterized and modified rock bream β-actin promoter. The obtained fragment was then subcloned into pGEM T easy vector and excised with XhoI and HindIII digestion and ligated to pBAcoreCArG(−)-EGFP digested with the same restriction enzyme. pβAcore R was designed to replace the putative transcrip- tion initiator element of β-actin promoter to that containing the BglII restriction enzyme site. we constructed EGFP expression vector in which EGFP coding sequence was put immediately after TSS of core promoter. 5′ rapid amplification of cDNA ends (RACE) primer positioned in second exon was designed. original direction) and pβAcoreCArG(−)-EGFP (4. In the previous study.1. Two oligonucleotides encoding shRNA sequence (200 μM each) were annealed by heating at 95°C for 3 min in annealing buffer (100 mM Tris–Cl (pH 7. 124–144) was reported previously (Zenke and Kim 2008).
we have constructed shRNA expression vector in which shRNA coding sequence was placed immediately next to TSS of core promoter. and R3 using pβA-EGFP as a template. R2. For this.Appl Microbiol Biotechnol (2010) 85:679–690 Table 1 Oligonucleotides used in the present study Name RbBA 5′RACE pβAcore F pβAcore R SV40mpA F SV40mpA R EGFP F EGFP R pBAcore-EGFP F1 pBAcore-EGFP F2 EGFP F2 EGFP R2 shEGFP-up shEGFP-bo pmβAd-shEGFP R1 pmβAd-shEGFP R2 pmβAd-shEGFP R3 pmβAd-shEGFPsc R1 pmβAd-shEGFPsc R2 pmβAd-shEGFPsc R3 pβAcore F pmβAd-shMCP R1 pmβAd-shMCP R2 pmβAd-shMCP R3 pmβAd-shMCPsc R1 pmβAd-shMCPsc R3 EGFP detection F EGFP detection R 18S F 18S R lacZ F lacZ R MCP F MCP R 084 F 084 R Sequence (5′ to 3′) TCCCTGTTGGCTTTGGGGTTCAGG GTGAGTGACGCTGGACCAATCAG AGATCTGAATGTGTAAGCTGTGGGCG AGATCTGCGAAGCTTAATAAAGGATCTTTTATTTTC ACTAGTCCAAGCTAGCGGCCGCATAC AAGCTTCGCCACCATGGTGAGCAAGG GCAGTGAAAAAAATGCTTTATTTGTG AAAAAACAAGCGCCCACAGCTTACACATTCAAT GGTGAGCAAGGGCGAG CTCGAGCCGGGCAGCTGACGCAGTATAAAAAAC AAGCGCCCAC GGATCCCGCCACCATGGTGAGCAAGG AAGCTTTTACTTGTACAGCTCGTCCATG GATCTGCTGACCCTGAAGTTCATCTTCAAGAGAG ATGAACTTCAGGGTCAGCTT AGCTAAGCTGACCCTGAAGTTCATCTCTCTTGAA GATGAACTTCAGGGTCAGCA TCTCTTGAAGATGAACTTCAGGGTCAGCTGAATG TGTAAGCTGTGGGC CATCTCTCTTGAAGATGAACTTC AAGCTTAAGCTGACCCTGAAGTTCATCTCTCTTG AAGATGA TCTCTTGAAGATGAACTTGTGGGTCAGCTGAATG TGTAAGCTGTGGGC CATCTCTCTTGAAGATGAACTTG AAGCTTAAGCTGACCCACAAGTTCATCTCTCTTG AAGATGA GTGAGTGACGCTGGACCAATCAG TCTCTTGAGAATTAGCATGGCCAGTCTGTGAATG TGTAAGCTGTGG AATTTCTCTTGAGAATTAGCATG AAGCTTAACAGACTGGCCATGCTAATTTCTCTTG AGAATTAG TCTCTTGAGAATTAGCATGCGCAGTCTGTGAATG TGTAAGCTGTGG AAGCTTAACAGACTGCGCATGCTAATTTCTCTTG AGAATTAG ATGGTGAGCAAGGGCGAGGA TGGTCGGGGTAGCGGCTGAAG TAGTTGGTGGAGCGATTTGTC CACTTGTCCCTCTAAGAAGTTG ACTATCCCGACCGCCTTACT TAGCGGCTGATGTTGAACTG ACAAGTGAGGAGCGTGAGGT GTCGAGATGCACCAAGGAAT GGGTCATGACACTGCAACAG GCCTGGTTGATTTCGTCAAT Purpose 681 5′ RACE of rock bream β-actin Cloning of core promoter Cloning of core promoter Cloning of SV40mpA Cloning of SV40mpA Cloning of EGFP Cloning of EGFP Construction of pβAcoreCArG(-)d-EGFP Construction of pβAcoreCArG(-)d-EGFP Cloning of EGFP for pCMVm-EGFP Cloning of EGFP for pCMVm-EGFP Construction of pmβA-shEGFP Construction of pmβA-shEGFP Construction of pmβAd-shEGFP Construction of pmβAd-shEGFP Construction of pmβAd-shEGFP Construction of pmβAd-shEGFP Construction of pmβAd-shEGFPsc Construction of pmβAd-shEGFPsc Construction of pmβAd Construction of pmβAd-shMCP Construction of pmβAd-shMCP Construction of pmβAd-shMCP Construction of pmβAd-shMCPsc Construction of pmβAd-shMCPsc Detection Detection Detection Detection Detection Detection Detection Detection Detection Detection of of of of of of of of of of EGFP EGFP 18s rRNA 18s rRNA lacZ lacZ MCP MCP ORF084 ORF084 BglII and HindIII. the part of core promoter and shRNA sequence was amplified by three-step PCR with pβAcore F and three overlapping reverse primer pmβAd-shEGFP R1. 3a). PCR product was then subcloned into pGEM T easy vector and excised by XhoI and HindIII digestion and ligated to pmβA digested with the same . Next. which was named pmβA-shEGFP (Fig.
The part of core promoter and shRNA sequence was amplified by three-step PCR with pβAcore F and three overlapping reverse primers pmβAd-shMCP R1. and the supernatant was collected. Briefly. A410 value was then converted to lacZ unit using standard curve generated from serial dilution series of β-galactosidase (Sigma) where one unit is defined as the activity which hydrolyzes 1. and R3. Cell and transfection GF cells were grown in L-15 medium (Sigma) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 25°C. 0.000×g for 10 min. Control vector which expresses shRNA with two substituted nucleotides (underlined) in target sequence (5′-AAGCT GACCCACAAGTTCATC-3′) was also constructed in the same way using pβAcore F and pmβAd-shEGFPsc R1.4). lacZ activity was measured using each cell lysate. For dualvector construction which simultaneously expresses shRNA and EGFP. Control vector which expresses shRNA with two substituted nucleotides (underlined) in target sequence (5′-AACAGACTGCG CATGCTAATT-3′) was also constructed in the same way using pβAcore F and pmβAd-shMCPsc R1.000×g for 10 min. 2 μl of 10 mM dNTP mix (TaKaRa). Construction of siRNA expression vector targeting rock bream iridovirus major capsid protein siRNA target sequence for rock bream iridovirus (RBIV) major capsid protein (MCP) gene (5′-AACAGACTGGC CATGCTAATT-3′. PCR primers used in the amplification of each target gene were summarized in Table 1. The quantity of plasmid used for transfection was provided in the “Results” section.1 M Tris–Cl (pH 7. For normalization of transfection efficiency.5 μg of DNase treated RNA was incubated with 0. which was named pmβAd-shEGFPsc.45-μm cellulose acetate membrane filter (Advantec). centrifuged at 8. and R3 using pβA-EGFP as a template. For each sample. 1 mM 2-mercaptoethanol. Quantification of EGFP expression level by fluorometer After fluorescent microscopic observation. EGFP expression cassette was excised from pβA-EGFP by AatII digestion and ligated to each vector digested with the same restriction enzyme. each transfected cell was lysed in 200 μl of 0. reaction was stopped by addition of 50-μl stop solution (1 M sodium carbonate). relative EGFP expression level of each sample was calculated using the following formula: fluorescent reading/lacZ activity unit/microgram protein Growth curve analysis Cells were detached from each well with TrypLE express reagent (Gibco). R2. The number of survival cells was counted using hemocytometer after staining with trypan blue (Sigma). PCR product was then subcloned into pGEM T easy vector and excised by XhoI and HindIII digestion and ligated to pmβA digested with the same enzyme. 10 TCID50/ml RBIV was used at 24 h after transfection. virus suspension was stored in aliquots with 50% glycerol at −80 °C until use. total protein quantity was also measured by bicinchoninic acid methods. 164–184) was that reported by Dang et al. Promega). 50 μl of cell lysate was mixed with equal volume of 2× ONPG solution (200 mM phosphate buffer (pH 7. and then the supernatant containing soluble fraction was collected. To obtain cDNA. and A410 was measured using microplate reader (Bio-Rad).0 μmol of o-nitrophenyl β-D-galactoside to onitrophenol and D-galactose per minute at pH 7. The homogenate was centrifuged at 2. filtered through 0.3 at 37 °C. and 1 mg/ml onitrophenyl β-D-galactoside) and incubated for 1 h at 37°C.5 μl of random primer (0.25 μl of RNase inhibitor (Promega) in a final reaction volume of 10 μl. Subsequently. which were named pβ A-EGFP-m β A-shEGFP. Virus RBIV was prepared from spleen of RBIV-infected rock bream. and 0. 2 mM MgCl2.1% Triton X-100 buffered with 0. Rock bream β-actin promoter-driven siRNA expression vector was constructed using three-step PCR approach described above.682 Appl Microbiol Biotechnol (2010) 85:679–690 enzyme. R2. and R3. Semiquantitative RT-PCR Total RNA was isolated from each group using RNAiso reagent (TaKaRa) and treated with RQ1 RNase-free DNase (Promega).5 μg/ml. respectively. Transfection of plasmid was carried out using Fugene6 (Roche) transfection reagent according to the manufacturer's instruction. which was named pmβAd-shEGFP (Fig. PCR was done in a 10-μl reaction containing 5 μl of 2× Prime Taq Premix (Genet . 3a). Spleens were homogenized in approximately equal volume of MEM (Sigma) with no supplement. The virus titer was determined using the 50% tissue culture infective dose (TCID50) method. Subsequently. which was named pmβAd-shMCP. p βA-EGFP-m βAdshEGFP. and pβA-EGFP-pmβAd-shEGFPsc.5 μl of MMLV reverse transcriptase (RT.4). R2. (2008) targeting MCP of red sea bream iridovirus. 0. After incubation. For virus inoculation. Promega) at 70 °C for 10 min and further incubated at 42 °C for 60 min in reaction mixture containing 2 μl of 5× reaction buffer. which was named pmβAd-shEGFPsc. which is very closely related virus to RBIV.
1). The amplification procedure included one cycle of 4 min at 95°C. Finally. and consensus sequence by Javahery et al. 1 μl of 10−1 diluted cDNA. and TATA box (TATAAA) were underlined. stained with EtBr. (1994) was shown above where Y is for T or C. EGFP expression vector pβAcore-EGFP which utilizes promoter with two modifications that is deletion of enhancer region and conversion of transcription initiator element to that containing BglII site exhibited about 50% lower transcriptional activity than pβA-EGFP which is driven by original β-actin promoter (Fig. Two CArG motifs were also found. however. and pictured using Gel Doc XR imaging device (Bio-Rad). 2c). we constructed pβAcoreCArG(−)d-EGFP in which the EGFP coding sequence was located immediately after TSS. PCR samples to be compared were electrophoresed on the same 1% agarose gel. 30 s at 72°C. we ligated the part of enhancer region containing CArG motif to upstream of β-actin core promoter. one is between TATA box and CAAT box (CCTTTTATGG). CArG motifs (CCTTTTATGG and CCTTA TATGG). Putative transcription initiation element was boxed. 30 s at 60°C. relative expression level was estimated by normalization with 18S rRNA and lacZ expression (for EGFP) or 18S rRNA (for RBIV MCP and ORF084). or 30 cycles (for RBIV MCP and ORF084) of 30 s at 95°C.Appl Microbiol Biotechnol (2010) 85:679–690 683 Bio).5 μM of each primer. with a final extension step of 7 min at 72°C. when the enhancer region put upstream in the opposite direction (pβAcoreCArG(−)EGFP). 0. Under fluorescence microscopy. βactin gene sequence and cDNA sequence obtained by 5′ RACE reaction was aligned. 25 cycles (for EGFP and lacZ). and distilled water. For semiquantitative analysis. Fig. both vectors showed the same high transcription level which was five times higher than that of pCMVm-EGFP driven by commercially available modified CMV promoter (Fig. 18 cycles (for 18S rRNA). we examined the transcriptional activity of core promoter of β -actin gene. Thus. and one is in first intron (CCTTATATGG. As a result. W is for A or G . CAAT box (CCAAT).600 bp upstream of ATG start codon. the intensity of each band of amplification product was measured using Quantity One software (Bio-Rad). we could observe EGFP fluorescence at 48 h after transfection to GF cells although it was much weaker than that observed in cells transfected with pβA-EGFP (data not shown). core promoter retained the original transcription activity compared to pβA-EGFP. First residue of this cDNA sequence was adenine residue and surrounded by putative translation initiation element TCACTCT where A is expected to be TSS. 2b). In the mRNA level. GF cells were transfected with dual- Results Sequence analysis and determination of TSS of rock bream β-actin gene The typical TATA box (TATAAA) and CAAT box (CCAAT) was found at about 1. Knockdown effect of siRNA expression vector driven by modified β-actin promoter In order to confirm the ability of modified β-actin promoter to express siRNA. Exon sequences were shown in uppercase. 1 The structure of rock bream β-actin gene promoter. Subsequently. Transcriptional activity of modified β-actin promoter Initially. and intron sequences were in lowercase. Fig. cDNA sequence of 497 bp obtained using 5′ RACE reaction was clearly matched to rock bream β-actin gene sequence.
3c). All data represented the mean of triplicate with standard deviation . pβAcoreEGFP (2).i. GF cells were also cotransfected with pFuguU6-shEGFP and pβA-EGFP. Furthermore. pβAcoreCArG(−)-EGFP. 4a—A. 4a–c.i.. While there was no difference in cell growth rate until the time of virus inoculation. EGFP expression level was quantified using fluorometer. As a result. which was characterized by the difference in the number of rounded and enlarged cells. E) than other cells transfected with pmβAd-shMCPsc (Fig. As a result. Growth curve analysis The difference in CPE among experimental groups was further examined by growth curve analysis of cells (Fig.684 Appl Microbiol Biotechnol (2010) 85:679–690 expression vector which simultaneously expresses EGFP and siRNA targeting EGFP. EGFP expression level was normalized against lacZ activity and total protein quantity of each sample. No obvious suppression effect was observed when cells were cotransfected with pmβAd-shEGFP and pβA-EGFP at the ratio of 1:1 and 1:3 or with pmβAd-shEGFPsc which express siRNA with two substitutions in target sequences. the space arising from the detachment of cells was obviously smaller in pm β Ad-shMCPtransfected cells than in other groups at 1 to 3 days p. cells transfected in advance with pmβAd-shMCP showed obviously less CPE through whole experimental period (Fig.i. a EGFP expression vectors constructed in this study was schematized. c Cells were cotransfected with 500 ng of pβA-lacZ and pβA-EGFP.3% reduction of EGFP expression when compared with control cells transfected with pβA-EGFP (Fig. (Fig. CPE observed after inoculation of RBIV After inoculation of RBIV following transfection of siRNA expression vector. pβAcoreCArG(+)-EGFP (3). 3b). C). figure of each construct is not in the scale.). 4b) When comparing experimental groups. At 48 h after transfection. 2 EGFP expression with modified rock bream β-actin promoter. Backbone of all vector presented is pGEM T easy vector (Promega) which comprises ColE1 origin of replication for amplification in host bacteria and ampicillin resistance gene for selection. b Cells were cotransfected with 500 ng of pβA-lacZ and pβA-EGFP (1). EGFP expression was reduced to 59. pβAcoreCArG(-)d-EGFP (5). (Fig. typical CPE characterized by rounding and enlargement of cells was observed in all experimental groups inoculated with RBIV while cells which received no inoculation formed a clear monolayer by that time (data not shown). B. Data represented mean of triplicate experiment with standard deviation. 3c). To compare the ability of modified β-actin promoter with fugu U6 promoter for expression of siRNA. there was about twice more vital cells observed in control cells without virus inoculation than all other groups inoculated with virus at Fig. G). F) or pmβAd-shEGFP (data not shown) or cells which received no transfection (Fig. or pβAcoreCArG(−)-EGFP (4). Furthermore. At 48 h after transfection. when cells were cotransfected with pmβAdshEGFP and pβA-EGFP at the ration of 5:1. For the clarity. or pCMVm-EGFP (6). only slight reduction of EGFP expression was observed in cells cotransfected with FuguU6shEGFP (Fig. Detailed procedure of vector construction was provided in “Materials and methods” section. Cells showing CPE continuously increased in whole experimental period and most of CPE showing cells were detached from cell monolayer as late as 5 days p. 4a—A. occurrence of CPE was monitored for up to 5 days postinoculation (p.2% of control cells transfected with pβA-EGFP only (Fig. EGFP expression level was quantified using a semiquantitative RT-PCR. As early as 1 day p. 4b). EGFP expression level was normalized against lacZ 18S rRNA. 4a–b. only cells transfected with p β A-EGFP-m β Ad-shEGFP showed 20.i.
Detailed procedure of vector construction was provided in “Materials and methods” section.Appl Microbiol Biotechnol (2010) 85:679–690 Fig. All data represented the mean of triplicate with standard deviation 685 1 day p. GF cells grown in 12-well cell culture plate were transfected with 500 ng of each dual-expression vector which simultaneously expresses siRNA and EGFP (b) or cotransfected with indicated quantity (μg) of siRNA expression vector and EGFP expression vector (c). Although expression of ORF084 gene was also analyzed as control.. All transfection included pβA-lacZ for normalization of transfection efficiency. At 2 and 3 days p.5 times more than in other groups. As a result.i. and total quantity of plasmid transfected to each well was adjusted to be equal with pβAcore-SV40mpA when needed. EGFP expression level was normalized against lacZ activity and total protein quantity of each sample. Semiquantitative analysis of viral mRNA expression Total RNA was isolated from cells at each time interval and subjected to expression analysis of viral mRNA. expression of MCP in pβAd-shMCP-transfected cells was reduced to about half of control up to 2 days p.i. This difference was further kept until 3 days p. the number of vital cells in pβAdshMCP-transfected ones was about 1. there was no difference in expression level among all groups and time intervals. . but not observed at 5 days p. and it was almost the same number as the control cells without inoculation. At 2 days p.i.i.i. 3 Knockdown of EGFP expression by siRNA expression vector. while there was no difference in MCP expression among other cell groups (Fig. a siRNA expression vectors constructed in this study were schematized.i. we could observe the difference in growth rate among cells transfected with each vector. in which the number of vital cells in all virus-inoculated cells was almost the same and about 80% less than control cells without infection... At 48 h after transfection. 4c). EGFP expression level was quantified using fluorometer.
and TATA box. 1990b). while transcription initiator element can enhance TATA box activity in the TATA-box-containing promoter (Weis and Reinberg 1992. 1990a). we were convinced that the adenine residue was actually a TSS of rock bream β-actin promoter. Transcription initiator element acts like TATA box and defines correct TSS in the promoter lacking TATA box. and infection was indicated by dashed arrow. H) were also included in the experiment. 3. G. which was revealed to be an adenine residue surrounded by putative translation initiation element TCACTCT (TSS is in bold). MCP. (1988) reported that deletion of enhancer region from human β-actin promoter reduced its transcriptional activity about 5% of wild-type promoter and further demonstrated that enhancer activity strongly depends on the presence of CArG motif. we have determined TSS of rock bream β-actin promoter using 5′ RACE technique. In order to optimize rock bream β-actin promoter for the expression of siRNA. shMCPsc. ORF084. 1984. CArG motif. 2002). pmβAd-shMCPsc (B. It is suggested that the transcription initiator element which overlaps TSS described as a loose consensus of (T/C)(T/C)AN(T/A)(T/C)(T/C) has two functions for transcription event. we could observe EGFP fluorescence at 48 h after transfection to GF cells although it was much weaker than that observed in cells transfected with pβA-EGFP or pβAcoreCArG(−)EGFP (data not shown). shEGFP. and 5 days (E.686 Appl Microbiol Biotechnol (2010) 85:679–690 Fig. 2. Furthermore. EGFP expression level was reduced to about 50% of cells transfected with EGFP expression vector driven by wildtype promoter. and 5 days postinoculation. pmβAd-shMCPsc. we then examined the necessity of enhancer region located downstream of TSS for full promoter activity since the siRNA coding sequence should be put immediately next to TSS. Under fluorescence microscopy. and low expression level observed under fluorescence microscopy should be due to the lack of kozak sequence in the construct. Sequence analysis revealed that rock bream β-actin gene sequence also had these core promoter elements and putative enhancer region containing CArG motif with the same arrangement of elements as in other β-actin promoters. shMCP. c Total RNA was isolated from cells inoculated with virus after transfection with each plasmid at 1. all of which are essential for full activity of β-actin promoter (Quitschke et al. C. transcriptional level of those vectors was equally high when compared with that of modified CMV promoter-driven pCMVm-EGFP. the core promoter retained transcriptional activity comparable to wild-type rock bream β-actin promoter. which indicates the importance of enhancer region also in rock bream β-actin promoter. 1990b). Number of survived cells was counted using trypan blue staining at each time point. it is suggested that mRNA should be correctly transcribed from TSS. However. Cells without transfection (C. (1994). As a result. it is known that the first intron containing second CArG motif shows enhancer activity (Liu et al. (1988) reported that 83-bp fragment containing CArG motif which was excised from human β-actin gene exhibited enhancer activity when placed upstream to enhancerless SV40 early promoter in either directions. This observation is consistent with previous studies where regulatory elements of β-actin gene as well as coding sequence are highly conserved beyond species (Ponte et al. Since this sequence clearly matches with the consensus transcription initiation element (T/C)(T/C)AN (T/A)(T/C)(T/C) reported by Javahery et al. Kawamoto et al. when GF cells were transfected with EGFP expression vector in which EGFP coding sequence was placed directly under the core promoter of rock bream β-actin and untranslated exon and first intron was completely deleted. Thus. O'Shea-Greenfield and Smale 1992). however. 1991). G) and cells without inoculation (D. Danilition et al. E). and it is suggested that overhangs in 5′ ends of shRNA should be minimized (Xia et al. Although it is b unknown why the enhancer activity was displayed only when the fragment was placed in opposite direction. Liu et al. It is possible that alternative transcription initiator element arose inside EGFP coding sequence in pβAcoreCArG(−)d-EGFP construct. 1989. b Cells were inoculated with RBIV at 24 h after transfection with each plasmid. and 18S rRNA genes were PCR-amplified from reversetranscribed cDNA samples using specific primers. β-actin promoter contains CAAT box. in order to examine the possible deletion of rock bream β-actin promoter transcription initiator element surrounding TSS. F. 3 days (A. B. which explains the comparable high transcriptional level. Finally. we constructed pβAcoreCArG(−)dEGFP in which the EGFP coding sequence was located immediately after TSS. pmβAd-shMCP. we next examined the enhancer activity of CArG motif contained in the first intron when it was placed upstream to the core promoter. H) postinoculation were shown. Liu et al. F). pmβAd-shEGFP. 1990b. a Cells were infected with RBIV at 24 h after transfection with pmβAdshMCP (A. when the enhancer region was placed upstream opposite to the original direction. Kawamoto et al. . Data were presented as mean of triplicate with standard deviation. 4 Inhibition of RBIV replication by siRNA expression vector. In the present study. The time of transfection was indicated by an arrow. Data were presented as mean value of triplicate relative expression normalized against 18S rRNA with standard deviation Discussion Generally. since we used detection primer complementary to 20 bp from ATG start codon and in this primer sequence there was no putative transcription initiator element matched to consensus existed. Furthermore. D). one similar observation was reported in carp that the activity of β-actin gene enhancer has both orientation and position dependence unlike other general enhancers (Liu et al. In the mRNA level. Representative pictures at 1 day.
Appl Microbiol Biotechnol (2010) 85:679–690 687 .
suggesting the establishment of RBIV infection in GF cells.i. Although virulence of RBIV is very high and the mortality of infected fish usually reaches 100%. these observations suggest that inhibition effect obtained with pmβAd-shMCP was highly specific. and at the ratio of either 1:3 or 1:1.. RBIV is the causative agent of iridoviridosis of rock bream. Since it was demonstrated in several studies that even short siRNA could induce nonspecific innate immune response in mammalian cells and fish cells (Schyth et al. Although it is possible to further modify the promoter to achieve . there was no inhibition effect in cells transfected with these control vectors. it is also possible that virus replication in control cells has naturally fallen down at late phase. we could observe the reduction of EGFP expression in dual-expression vector transfection experiment only when shRNA coding sequence targeting EGFP was placed immediately next to TSS. the result is the successful induction of RNAi in host cells. several studies have reported the successful use of siRNA to inhibit virus replication in vitro (Schyth et al. 2009). 2008. 2002. and there was no effect in other control siRNA with scrambled or unrelated sequences.i. Jun et al. In the present study. we observed the same characteristic of CPE in cells inoculated with RBIV. 2006). In addition to these hypotheses. In fish species. which was evidenced by the appearance of less CPE and higher growth rate and the reduction of MCP gene expression confirmed by RT-PCR analysis. thus. Ma et al. Dang et al. (2008) in which siRNA with the same sequence as used in the present study specifically inhibited RSIV replication. 2007). It is known that CPE caused by fish iridovirus is characterized by rounding and enlargement of cells resulting from cell apoptosis (Imajoh et al. These observations suggest that inhibitory effect of siRNA expressed from plasmid vector might not be long lasting and/or virus replication level might be much more active than inhibitory effect of siRNA at the late phase of virus infection. 2008. cell growth rate fell to the same level as control cells at 5 days p. rose to the same level as the cells transfected with pmβAdshMCP. One promising application of RNAi is the therapeutic use against virus infection (Tan and Yin 2004. 2004. since the first use of siRNA for tiger frog virus infection in fathead minnow cell line (Xie et al. Svoboda 2007). This result indicated the importance of proximality of shRNA coding sequence to TSS as suggested in other studies (Xia et al. GF cells which is very susceptible to RBIV infection were inoculated with RBIV after transfection with siRNA expression vector. 2007. There was no reduction of EGFP expression observed in control groups where cells were transfected with vector which express siRNA targeting EGFP with two sequence substitution.688 Appl Microbiol Biotechnol (2010) 85:679–690 Based on the characterization and modification of rock bream β-actin promoter. our observation seems to be the result of complicated interaction of virus replication and inhibitory effect of siRNA though it is at least obvious that siRNA was successfully expressed from vector and degraded viral mRNA. 2006. The relatively low inhibition effect observed in the cotransfection experiment was supposed to be due to high background from cells only transfected with EGFP expression vector since the transfection efficiency of GF cells is low as reported in previous study (Zenke and Kim 2008). When comparing difference in CPE. This result is consistent with Dang et al. Furthermore. which has severely damaged rock bream aquaculture industry in South Korea. there was no inhibition effect observed. Although we could not directly confirm the activation of innate immune system of GF cells because of the lack of information about type I interferon or immune-related genes of GF cells. the suppression level was slightly higher than that observed in cells transfected with fugu U6 promoter-driven siRNA expression construct. Furthermore. Huang et al. we included control siRNA expression vectors which express sequence substituted siRNA (pmBAd-shMCPsc) or siRNA with completely different sequence (pmBAd-shEGFP). In conclusion. although inhibition of RBIV replication seemed to be kept for whole experimental period in the morphological observation level. 2009).3% against control was obtained when the ratio of EGFP expression vector versus siRNA expression vector was 1:5. In cotransfection experiment. 2005). the critical methods for treating or preventing RBIV infection has not yet been reported (Jeong et al. RT-PCR analysis revealed that reduction of viral protein mRNAs observed in cells transfected with pmβAd-shMCP are observed only in MCP but not in unrelated ORF084. and the highest reduction of about 59. GF cells transfected with pmβAd-shMCP were shown to be more resistant than control groups. 2007). To examine the possible use of siRNA expression vector for the inhibition of RBIV replication in vitro. Thinking together. As a result. Ruiz et al. 2004. we have constructed modified β-actin promoter-driven siRNA expression vector and demonstrated that rock bream β-actin promoter seems to have more potential than U6 promoter for use in fish species. thus. 2006.i. siRNA expression vector could reduce EGFP expression dose-dependently. Haasnoot et al. where shRNA coding sequence was placed immediately next to transcription initiator element or TSS. This dose dependency was also true for pFuguU6shEGFP although reduction of EGFP expression level was only about 80% at maximum. and the result of RT-PCR analysis showed that MCP gene expression was reduced only up to 2 days p. we constructed two types of siRNA expression vector targeting EGFP. the number of vital cells in control groups at 5 days p. However. As a result of virus inoculation. Song et al.
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