AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

甘蓝型油菜三种种子储藏蛋白和丙酮酸羧化酶基因片段的克隆及hpRNA表达载体的构建

利用PCR从甘蓝型油菜(Brassica napusL.)华双4号基因组DNA中扩增出种子储藏蛋白cruc iferin、nap-in、oleosin和丙酮酸羧化酶(PEPC)基因片段,再以扩增出的片段为模板设计引物从一端扩增出4个相应的小片段,然后将同一基因的大小两个片段反向连接,插入到种子特异表达载体2300-nap多克隆位点的nap in启动子和nos终止子之间,构建成可以在油菜种子中转录表达发夹RNA(Hairp in RNA,hpRNA)结构的植物表达载体。

甘蓝型油菜依赖焦磷酸的磷酸果糖激酶(PFK)基因片段克隆及hpRNAi载体构建

依赖焦磷酸的磷酸果糖激酶(PFK)是糖酵解途径的关键酶。本研究首先构建带有种子特异性napin启动子和Nos终止子的植物表达载体2300-nap及带有组成型35S启动子和Nos终止子的植物表达载体2300-35S;然后用PCR法从甘蓝型油菜(BrassicanapusL.)中油119总基因组中扩增出依赖焦磷酸的磷酸果糖激酶(PFK)基因片段,再以扩增出的PFK基因片段作模板设计引物扩增出一个相应的小片段。将两个PFK基因片段反向连接,插入到植物表达载体2300-nap的napin启动子和nos终止子之间,植物表达载体2300-35S的35S启动子和nos终止子之间,分别构建成可转录表达出发夹RNA(hairpinRNA,hpRNA)结构的种子特异型和组成型油菜RNA干扰载体,为今后油菜利用RNA干扰(RNAi)提高含油量的基因工程研究奠定了基础。

甘蓝型油菜乙醇酸氧化酶(GO)基因片段的克隆及hpRNA表达载体构建

通过PCR从甘蓝型油菜(Brassica napus)华双4号基因组DNA中扩增出乙醇酸氧化酶(GO)基因片段,DNA序列分析表明扩增GO基因片段的外显子部分与报道序列相同.以扩增出的GO基因片段作模板从一端设计引物扩增出一个相应的小片段.将GO基因大小两个片段反向连接,插入到植物表达载体2300-nap的napin启动子和nos终止子之间,植物表达载体2300-35S的35S启动子和nos终止子之间,分别构建成可转录表达出发夹RNA(hairpin RNA,hpRNA)结构的种子特异型和组成型油菜hpRNA干扰载体.

利用ihpRNA介导的基因沉默培育抗冷糖化马铃薯

为培育抗冷糖化的转基因马铃薯材料,克隆了368 bp的马铃薯酸性转化酶(AcInv)基因3′端非翻译区(3′UTR)和281 bp的马铃薯VP1-ABI3-like protein基因的第一内含子,构建了内含子连接的马铃薯AcInv基因3′UTR的发夹型RNA(ihpRNA)载体pBIV;采用微束激光穿刺技术转化马铃薯品种,得到了149个转基因株系,其中139个转基因株系在低温贮藏35 d时块茎还原糖含量低于亲本材料。RT-PCR分析表明还原糖含量降低的转基因植株中检测不到AcInv基因mRNA的积累。本研究结果表明以3′UTR作为RNAi载体的干涉片段可以有效地抑制靶标基因mRNA的积累,通过对AcInv沉默可获得抗低温糖化的马铃薯育种材料。

甘蓝型油菜种子中G3PDH基因特异性表达的ihpRNA载体构建

为验证甘蓝型油菜甘油-3-磷酸脱氢酶基因(G3PDH)的功能,采用PCR的方法克隆BnG3PDH557bp的干扰靶序列,将其正向片段插入到中间载体pHurricane的NotI酶切位点、反向重复序列插入到XhoI酶切位点,构建ihpRNA载体反向重复框,BamHI、EcoRI酶切下反向重复框后组装到由种子特异性表达的napin启动子驱动的pCAMBIAl390植物表达载体上。结果表明:PCR扩增产物、限制性内切酶酶切产物与预期片段长度相符。其中,1.7kb的片段包括正向干扰靶片段和部分AtFad2内含子序列,2.3kb大小的片段是干扰反向重复框。种子特异性表达的BnG3PDHihpRNA载体成功构建。

甘蓝型油菜和Lesquerella fendleri fad2基因片段克隆及hpRNA植物表达载体的构建

首先利用PCR从甘蓝型油菜(Brassica napus L.)和Lesquerella fendleri基因组DNA中分别扩增出Δ12-脂肪酸去饱和酶fad2基因片段,再以扩增出的片段为模板设计引物从一端扩增出相应的小片段,然后将同一基因的大小片段反向连接,插入到种子特异表达载体2300-nap多克隆位点的napin启动子和nos终止子之间,构建成可以在种子中转录表达发夹RNA(Hairpin RNA,hpRNA)结构的植物表达载体.

番木瓜eIF4E和eIFiso4E基因嵌合hpRNA载体一步快速构建及沉默效果的研究

番木瓜环斑病毒(Papaya ringspot virus,PRSV)严重威胁番木瓜种植业的发展,且目前没有十分有效的防治办法。病毒侵染植物依赖寄主因子的协助,真核翻译起始因子4E(eukaryotic initiation factor 4E,eIF4E)是多种RNA病毒侵染植物的必需因子。以番木瓜eIF4E家族基因为研究对象,构建同时干扰其eIF4E和eIFiso4E基因的发卡RNA(hairpin RNA,hpRNA)载体,并将其导入到番木瓜叶肉原生质中。通过荧光实时定量检测发现,番木瓜中eIF4E和eIFiso4E基因的表达量分别下降了49.8%和67.6%,这为进一步研究番木瓜eIF4E家族基因对PRSV侵染的影响以及利用RNA干扰技术靶向植物基因的病毒防治新策略提供理论和实践依据。

实时定量PCR分析PtDr101基因hpRNA干扰下的表达

PtDr101基因从三倍体毛白杨(Popu1us tomentosa× P. bolleana)×P.tomentosa]中克隆获得,能够编码NBS-LRR型蛋白,受到水杨酸和甲基茉莉酸诱导表达,是一种广谱的抗病基因。本研究利用实时定量PCR技术分析转PtDr101基因hpRNA干扰载体的表达模式,分析发现4个表达模式显著差异的转基因株系,为进一步功能鉴定提供材料。本研究表明实时定量PCR能够高灵敏度、精确地检测hpRNA干扰情况下的基因表达量。

Growth inhibition induced by short hairpin RNA to silence survivin gene in human pancreatic cancer cells

BACKGROUND: Survivin is known to be overexpressed in various human malignancies, including pancreatic cancer, and mediates cancer cell proliferation and tumor growth, so the regulation of this molecule could be a new strategy for treating pancreatic cancer. In this study, short hairpin RNAs (shRNAs) specific to survivin were introduced into human pancreatic cancer Patu8988 cells to investigate the inhibitory effects on survivin expression and cell proliferation in vitro and in vivo. METHODS: Three kinds of shRNA specific to the survivin gene were designed and cloned into eukaryotic expression plasmid pGenesil-1 vector. Subsequently the recombinant plasmids were transfected into human pancreatic cancer Patu8988 cells with lipfectamine (TM) 2000 reagent. The mRNA and protein expressions of survivin in the transiently transfected Patu8988 cells were determined by RT-PCR, flow cytometry, and Western blotting analysis. The proliferation inhibition rates of stably transfected Patu8988 cells were determined by MTT assay. The antitumor activities of the three kinds of survivin-shRNA plasmids were evaluated in BALB/c nude mice inoculated with Patu8988 cells and bearing human pancreatic cancer. RESULTS: The three survivin-shRNA plasmids named pGenesil-1-survivin-1, pGenesil-1-survivin-2 and pGenesil-1-survivin-1+2 (with double interfering RNA sites) were successfully constructed, and were confirmed by restriction enzyme cutting and sequencing. At 48 hours after transfection, the expression of survivin mRNA and protein was inhibited in Patu8988 cells transfected with pGenesil-1-survivin-1, pGenesil-1-survivin-2, and pGenesil-1-survivin-1+2 when compared with that of either pGenesil-1-NC (with scrambled small interfering RNA) transfected cells or control cells (P<0.05). The MTT results showed that the proliferation rates of Patu8988 cells stably transfected with survivin-shRNA plasmids were reduced when compared with that of either pGenesil-1-NC transfected cells or control cells (P<0.01). Furthermore, when Patu8988 cells stably transfected with survivin-shRNA were injected into BALB/c nude mice, tumor growth was dramatically lower and the tumor was smaller than that of either pGenesil-1-NC transfected cells or control cells (P<0.01). The inhibitory effect of pGenesil-1-survivin-1 was the best among the three kinds of survivin-shRNA plasmids, but no combination of inhibitory effects was found in pGenesil-1-survivin-1+2. CONCLUSIONS: shRNAs specific to survivin have gene silencing effects and inhibit pancreatic cancer cell proliferation. shRNA activity against survivin could be of potential value in gene therapy for pancreatic cancer. However, shRNAs with double combining sites did not significantly enhance the interference compared with single site shRNAs, therefore further studies on this are needed.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Activity of Rat Vascular Smooth Muscle Cells

To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 （PG1）, pGenesil-1/OPNshRNA2 （PG2） and pGenesil-1/OPNshRNAHK （PGH） were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% （P〈0.01） by PG1, 73% and 52% （P〈0.01） by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type Ⅰ and Ⅲ collagen. It is concluded that recombinant plasmid can be success-fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention （PCI） by RNA interference （RNAi） technology.

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

Short hairpin RNA-mediated knockdown of nuclear factor erythroid 2-like 3 exhibits tumor-suppressing effects in hepatocellular carcinoma cells

BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n' collar basic-region leucine zipper family of transcription factors. NFE2 L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.AIM To explore the expression and biological function of NFE2 L3 in HCC.METHODS We analyzed the expression of NFE2 L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas(TCGA) data portal. Short hairpin RNA(shRNA) interference technology was utilized to knock down NFE2 L3 in vitro. Cell apoptosis, clone formation, proliferation, migration,and invasion assays were used to identify the biological effects of NFE2 L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition(EMT) markers was examined by Western blot analysis.RESULTS TCGA analysis showed that NFE2 L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2 L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2 L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2 L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.CONCLUSION Our study showed that shRNA-mediated knockdown of NFE2 L3 exhibited tumor-suppressing effects in HCC cells.

Short Hairpin RNA-mediated MDR1 Gene Silencing Increases Apoptosis of Human Ovarian Cancer Cell Line A2780/Taxol

Objective： Recurrent ovarian cancer is often resistant to drugs such as paclitaxel. Short hairpin RNA （shRNA） targeting MDRI, a gene involved in the process of drug resistance, may be a promising strategy to overcome drug resistance. Methods： Construction and identification of eukaryotic expression plasmid was transiently transfected into human ovarian cancer ce plasmid of shRNA targeting on MDR1 gene. The ne A2780/Taxol. Apoptosis was determined by flow cytometry using annexin V-FITC/PI double labeling. Expression of MDRI mRNA was detected by quantitative polymerase chain reaction （qPCR） and P-glycoprotein expression was detected using Western blot. Results The IC50 of paclitaxel in MDR1 shRNA-transfected group was significantly reduced （1.986±0.153）μmol/ml as compared with that in negative control （5.246±0.107）μmol/ml and empty vector-transfected group （5.212±0.075）μmol/ml （P〈0.05）. The percent of the relative reverse sensitivity to paclitaxel on A2780/Taxol cells was 67.1%, and the apoptotic rate was significantly increased [（6.977±0.333）%] compared with control [（1.637±0.111）%] and empty vector-transfected group [（1.663±0.114）%] （P〈0.05）. Expressions of MDR1 mRNA and P-glycoprotein were significantly reduced compared with control （P〈0.05）. Conclusion： The present study demonstrated that the eukaryotic expression plasmid of shRNA targeting on MDRI inhibited the expression of MDRI effectively, thus enhance the sensitivity of A2780/Taxol cells to paclitaxel.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Invasion of Human Renal Cancer Cells

The influence of short hairpin RNA(shRNA)-mediated osteopontin(OPN)gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated.Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids,which were transferred into the cultured ACHN cells by LipofectamineTM 2000.The cells transfected by shRNA expression vectors(ACHN/OPN)were visualized under an inverted microscope and screened...

Complement factor B knockdown by short hairpin RNA inhibits laser-induced choroidal neovascularization in rats

AIM:To evaluate whether recombinant complement factor B(CFB)short hairpin RNA(sh RNA)reduces laserinduced choroidal neovascularization(CNV)in rats.METHODS:Laser-induced rat CNV model was established,and then the animals underwent fundus fluorescence angiography(FFA)and hematoxylin and eosin(HE)staining.On day 3 and 7 after photocoagulation,the expression of CFB and membrane attack complex(MAC)was detected by immunhischemistry.A recombinant CFBsh RNA plasmid was constructed.CFB and scrambled sh RNA plasmids were intravenous injected into rats via the tail vein on the day of laser treatment,respectively.On day 7,the incidence of CNV was determined by FFA,and the expression of CFB and vascular endothelial growth factor(VEGF)in retinal pigment epithelium(RPE)/choroidal tissues was detected by immunhischemistry,Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)in CFB and scrambled sh RNA groups.The possible adverse effects of CFB-sh RNA injection were assessed by transmission electron microscopy and electroretinography.RESULTS:FFA and HE results indicated that a laserinduced rat CNV model was successfully established on day 7 after photocoagulation.The expression of CFB and MAC was extremely weak in normal retina and choroid,and increased on day 3 after photocoagulation.However,it started to reduce on day 7.CFB sh RNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues.FFA showed CFB knockdown significantly inhibited incidence of CNV in rats.Moreover,CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues.CFB sh RNA caused no obvious side effects in eyes.CONCLUSION:CFB knockdown significantly inhibits the formation and development of CNV in vivo through reducing the expression of VEGF,which is a potential therapy target.The alternative pathway of complement activation plays an important role in CNV formation.

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A

BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis has revealed high expression of centromere protein K(CENPK)in CRC.However,the role of CENPK in the progression of CRC is not well characterized.AIM To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A(CUL4A)in RKO and HCT116 cells.METHODS Human colon cancer samples were collected and tested using a human gene expression chip.We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis.In vitro experiments verified the function of this gene.We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction(qPCR),western blot,and flow cytometry.The effect of short hairpin RNA(shRNA)virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging.To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells,we performed a series of in vitro experiments,using qPCR,western blot,MTT assay,and flow cytometry.RESULTS We demonstrated overexpression of CENPK in human colon cancer samples.CENPK was an independent risk factor in patients with CRC.The downstream genes FBX32,CUL4A,and Yesassociated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells.Significantly delayed xenograft tumor emergence,slower growth rate,and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group.The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo.The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells,with overexpression of the CUL4A.CONCLUSION We indicated a potential role of CENPK in promoting tumor proliferation,and it may be a novel diagnostic and prognostic biomarker for CRC.

Small hairpin RNA against Bcl-2 increases MTX-induced apoptosis in Raji cells

Objective:The aim of this study was to study the effect of Bcl-2 small hairpin RNA(shRNA) enhancing methotrexate(MTX)-induced apoptosis of Raji cells.Methods:Expression plasmid with Bcl-2 shRNA was transfected into Raji cells by Lipofectmine 2000 and then treated with MTX.At 48 h of transfection,the expression level of Bcl-2 mRNA and protein was evaluated by RT-PCR and immunofluorescence.MTT assay was used to analyze cell proliferation at 24,48 and 72 h.Apoptosis was detected by Giemsa staining and flow cytometric cell cycle analysis.Results:After transfection with Bcl-2 shRNA,the expression levels of Bcl-2 mRNA and protein in Raji cells decreased(P < 0.05).Using Giemsa staining,cells transfected with Bcl-2 shRNA combined with MTX at 48 h displayed changes of apoptosis.MTX significantly inhibited the growth of cells after transfected with Bcl-2 shRNA(P < 0.05).Apoptotic rates of the Raji cells treated with Bcl-2 shRNA combined with MTX significantly increased(P < 0.05),compared with either control shRNA/MTX combination or MTX-treatment cells alone.Conclusion:Our results suggest the shRNA against Bcl-2 mRNA could increase MTX-induced apoptosis of Raji cells.

Construction of Short Hairpin RNA Vector with σNS & σC Genes of Avian Reovirus and Determination of Interference Effect

[ Objective] The aim was to explore novel method for treatment of Avian Reovirus. [ Method] According to the design principle of siRNA target sequences, siRNA templates were designed and synthesized and then cloned into the shRNA expression vector, namely, pSilencer-CMV 4.1 neo. Short hairpin RNA vector C1, C2, C3, which contain σC gene, and shRNA vector NS1, NS2, NS3, which contain aNS gene, were constructed separately. The constructed shRNA vectors and negative control were co-transfected into DF-1 cells with the eukaryotic expression vector pEG- FP-σC and pEGFP-σNS, respectively. [ Result] Observation through fluorescence microscope indicated that the constructed 6 shRNA could inhibit the expression of fusion protein to different degrees. In addition, results of Real-time PCR suggested that C3 and NS1 have the best interference effect to the viral duplication in vitro. [ Conclusionl Construction and selection of specific shRNA expression vectors inhibiting Avian Reovirus are significant for researching effects of σC and oNS proteins in infection and duplication of ARV, providing new idea for ARV antiviral therapy.

Suppression of Replication of Rabies Virus by Short Hairpin RNAs Expressed by Plasmid

Targeting the N gene of rabies virus (RV), four shRNA expression plasmids were designed and constructed based on the vector pRNATU6.3-Hygro that expresses fusion protein with GFP as a reporter gene. Four cell strains (N1, N2, N3, N4) expressing the short hairpin RNAs (shRNA) were obtained after the plasmids were transfected into BHK-21 cells and screened under the pressure of Hygromycin B (300 μg/mL). These cell strains were infected with 100× the TCID 50 of rabies virus CVS-11 strain, and the viral replication was quantified at 24, 48, 72 and 96 hours by directed immunofluorescence assay (DFA), real-time PCR, and the 50% tissue culture infective dose (TCID 50 ). The results showed variable inhibition of viral replication, with BHK-N2 being the most effective strain (99% inhibition). There was close correspondence between results using the three methods of evaluation. The shRNA-mediated inhibition persisted to at least 96 hours after infection. Effective inhibition of replication of RV in BHK-21 cells was achieved by siRNA targeting the N gene, with N 2 , aimed at the region starting at position 701 of the gene, being the most potent.