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双语推荐：RNAi载体

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构建针对M2型丙酮酸激酶(PKM2)基因3个可干扰序列设计小发夹RNA(small hairpin RNA,shRNA)载体,为下一步探索Ad-PKM2介导PKM2 RNAi对乳腺癌细胞作用及机制建立基础。方法:根据RNAi设计软件设计并合成3对shRNA模板序列,依次将它们克隆至pGenesil载体中构建重组质粒,通过MTT检测筛选出对乳腺癌细胞株MCF-7增殖效应影响最强的RNAi序列。结果:酶切鉴定和测序结果证实RNAi载体构建成功,均能高效感染MCF-7并抑制MCF-7细胞的增殖,pGenesil-PKM2-(1+2+3)的抑制能力最强。结论:针对PKM2多靶点RNAi载体成功构建,为下一步研究PKM2被沉默后细胞生物学行为的变化打下基础。
Objectiye:To construct a small hairpin RNA(shRNA)expression vector,mediated by recombinant adenovirus and targeting PKM2,and which will help for exploring the biological behavior change of breast cancer cells by means of knockingdown PKM2. Methods:Three pairs of templates of PKM2 shRNA were synthesized and cloned into the pGenesil system successfully. Then its effect on the proliferation of MCF-7 was detected by MTT. Results:pGenesil-PKM2 was con-structed successfully,and also it repressed proliferation of MCF-7 significantly after being transfected into the cells. Con-clusion:The construction of pGenesil-PKM2 paved the way for the further research.

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植物线粒体基因缺陷是细胞质雄性不育的主要原因。为了获得苎麻atp6和atp9基因RNA干扰（RNAi）表达载体，根据已报道的苎麻atp6和atp9基因序列设计引物，利用RT-PCR克隆了atp6和atp9基因的部分cDNA片段，将目的基因正反向片段连接入RNAi载体pHANNIBAL，再将其表达框连入表达载体pCAMBIA 1300。结果表明，所克隆的cDNA片段经序列比对后确认为目的基因片段，经酶切和测序验证确认完成了atp6和atp9基因RNAi载体pCAM-6SR和pCAM-9SR的构建。atp6和atp9基因RNAi载体是验证苎麻雄性不育的基础，也为苎麻遗传工程改良奠定了技术基础。
Defects in mitochondrial genes are the main sources of cytoplasmic male sterility in plants. In order to construct RNAi vectors of atp6 and atp9 genes, this article designed primers and cloned cDNA fragments of atp6 and atp9 genes by RT-PCR according to the reported gene sequences in ramie. Sense and reverse fragments of the genes were introduced into RNAi vector pHANNIBAL, and then the expression frame was cloned into pCAMBIA 1300. The alignment results showed that the cloned cDNA were target gene fragments. RNAi expression vectors pCAM-6SR and pCAM-9SR were confirmed by restriction enzyme and sequencing. Construction of atp6 and atp9 RNAi vectors was the foundation of validating their function in male sterility, and this work also laid the technical foundation of genetic improvement in ramie.

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观察转化生长因子β-R2特异性短发夹RNA(shRNA)对人晶状体上皮细胞TGFβ-R2表达的影响。方法:根据小干扰RNA(siRNA)的设计原则,针对转化生长因子β-受体2基因序列特征构建特异性shRNA(RNAi)载体,与TGFβ-R2过表达载体共转染293T细胞,经Western blot筛选出有效RNAi载体后,进行扩增、纯化、滴度测定,并转染培养的人LECs,qPCR检测TGFβ-R2siRNA慢病毒载体对TGFβ-R2 mRNA表达的影响。结果:经Western blot筛选,TGFβ-R2/GV1 15RNAi#1对目的基因的表达敲减效果最明显,慢病毒包装,滴度为8E+8TU/mL,感染人LECs细胞后,对TGFβ-R2基因有显著的敲减效果,达到78.1%(P0.05)。结论:TGFβ-R2 RNAi载体构建成功,并且能抑制人晶状体上皮细胞TGFβ-R2 mRNA的表达。
AIM:To observe the suppressing effect of specific small hairpin RNA ( shRNA) on TGFβ-R2 expression in human lens epithelial cells ( LECs) . METHODS: Specific shRNA expression vector was constructed according to the design principles of TGFβ-R2 mRNA human GeneBank siRNA synthesis and transfected into cultured 293T with TGFβ-R2 over expression vector. After effective RNAi vector was selected by Western blot, then amplification, purification, and titration.lentivirus-TGF beta-R2 shRNA was transfected into LECs, then TGFβ-R2 mRNA expression in these cells was detected by the real -time fluorescence quantitative PCR detecting system ( qPCR) . RESULTS: TGFβ-R2/GV115RNAi # 1 target gene expression knockdown effect is the most obvious;the titer of packaging lentivirus is 8E+8 TU/mL; the knockdown effect is significant, the interfering efficiency is 78.1%(P CONCLUSION: TGFβ-R2 RNAi vector was successfully constructed, and can inhibit the expression of TGFβ-R2 mRNA in human LECs.

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采用基因芯片技术，从油菜中鉴定了一个油分积累优势表达基因即丙酮酸激酶（Pyruvate kinase, PK）基因。为构建甘蓝型油菜PK基因的RNA干扰(RNAi)载体，通过设计引物克隆了 PK基因长498 bp的 siRNA靶序列，连接到 pEASY-T1载体上，采用 NotI和XhoI酶切，酶切产物连接到本课题组新近改造的 RNAi 平台载体 pHurricane 的 NotI 和XhoI位点，通过多重 PCR 鉴定证实载体构建成功，然后将上述 PK基因反向重复框克隆到加入 napin启动子的 pCAMBIA1390表达载体相应的酶切位点上，构建具有种子特异性表达的PK基因的RNAi载体。限制性内切酶酶切证实载体构建成功，为进一步研究 PK基因参与决定油菜油分积累的分子机理和代谢调控奠定了基础。
A predominantly expressed gene, pyruvate kinease (PK) gene, control ing oil accumulation, had been identified in previous study. To construct a PK RNAi vector, a 498-bp target PK gene sequence was amplified and transferred into the pEASY-T1vector. Subsequently, the target DNA fragments were cut off by enzymes Not I and Xho I and directional y inserted into plant RNAi platform vector pHurricane, a newly developed RNAi vector in our lab, to form the PK inverse repeats. The PK inverted repeats fragment was then cloned into a modified vector pCAMBIA1390, driven by a rapeseed seed-specific promoter napin. Restriction enzyme digestion verified the successful construction of RNA interference vector. The PK RNAi con-struction wil lay a foundation for study in the function of PK in oil accumulation and metabolism in rapeseeds.

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FADD(Fas-associated death domain protein)存在于Fas/FasL系统中,是信号传导通路的一个信号连接蛋白,FADD通过传递凋亡信号,从而介导细胞凋亡。为进一步验证FADD基因抑制细胞增殖和促进细胞凋亡的作用,从牛卵巢组织中扩增FADD基因,将FADD基因连接到带有绿色荧光蛋白报告基因的真核表达载体pAcGFP-N1中,构建过表达FADD基因载体,并构建FADD基因的RNAi载体;用脂质体介导法将FADD基因RNAi载体、真核表达载体转染到牛胎儿成纤维细胞中,观察有无荧光的表达,并使用Real-Time qPCR和Western blot方法检测FADD基因mRNA、蛋白水平的表达情况。结果表明:成功构建出FADD基因RNAi载体和高表达载体,重组质粒转染牛胎儿成纤维细胞24 h后在荧光显微镜下可观察到绿色荧光,转染效率可达50%。
Fas-associated death domain protein(FADD)is connected to a signal protein signaling pathway in Fas/FasL system which mediates apoptosis guide by passing apoptotic signals .To further verify the function of FADD gene in inhibiting cell proliferation and promoting apoptosis,we cloned FADD gene in bovine ovary tissue with molecular cloning technique,directionally cloned the amplified FADD gene into eukaryotic expression vector pAcGFP-Nl including AcGFP and constructed the fusion protein recombinant plasmid .Using gene-silencing technology,we constructed RNA interference(RNAi)vector .And then we transfected pAcGFP-N1-FADD and RNAi into bovine fetal fibroblasts(BEF)cell mediated by Lipofec-tamine 2000,observed the expression of AcGFP and detected the mRNA and protein level of FADD by Real-Time qPCR and Western blot .The results showed that cattle FADD gene was successfully cloned, and RNAi vectors and pAcGFP-N1-FADD fusion protein eukaryotic expression vector were successfully constructed .

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水稻黑条矮缩病毒（Rice black-streaked dwarf virus,RBSDV）不同双链RNA片段的三价RNAi（RNA interference）表达载体在转基因水稻中的研究尚未见报道。本研究以RBSDV已报道不同双链RNA序列为基础,设计了针对S2、S6、S10三个基因共605 bp的RNAi靶序列,通过基因合成的方法获得相应的目的片段。利用Gateway LR Clonase~TM Ⅱ Enzyme Mix的LR反应将目的基因构建到RNAi载体pBDL03中,然后通过农杆菌转化法转到水稻品种泰粳394中。通过PCR和荧光验证共获得45株阳性苗。T1代植株RBSDV抗性鉴定结果证实针对S2、S6和S10基因的三价RNA沉默载体对RBSDV具有良好的抗性。本研究结果为利用RNAi技术进行植物抗病毒研究奠定了基础。
Research on the trivalent RNAi (RNA interference) expression vector of different double-stranded RNA fragments of the Rice black-streaked dwarf virus (RBSDV) in transgenic rice has not been reported. In our study, a 605 bp RNAi target gene fragment for S2, S6, S10 genes was designed according to the published RBSDV genomic sequences and obtained by the gene synthesis method. This interference fragment was built into the pBDL03 RNA interference vector by LR reaction according to the Gateway? LR ClonaseTMIIEnzyme Mix Kit protocol and was then transformed into Rice Taijing 394 mediated by Agrobacterium. 45 transgenic rices were obtained by PCR and fluorescence detection. The results of T1 generation rices against RBSDV further illustrate that trivalent RNA silencing expression vectors targeting S2, S6, S10 genes can significantly improve the resis-tance against RBSDV. This work provided a basis of application of RNAi in transgenic plant resistance to viruses.

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目的：探讨心肌细胞JP2与SK2通道的相互作用。方法：构建靶向小鼠JP2基因的siRNA慢病毒载体（LV－JP2－RNAi－1、LV－JP2－RNAi－2），将其转染至小鼠心肌细胞，以转染空载体（LV－NC－EGFP）和未转染细胞（Ctrl－NC）作对照。采用实时荧光定量PCR和Western blot法分别检测各组心肌细胞JP2 mRNA和SK2蛋白的表达。结果：转染48 h后，各组小鼠心肌细胞JP2 mRNA表达的差异有统计学意义（F＝31．775，P＜0．001），LV－JP2－RNAi－1、LV－JP2－RNAi－2组较Ctrl－NC及LV－NC－EGFP组细胞降低。小鼠心肌细胞感染LV－JP2－RNAi－272 h后，SK2通道蛋白表达被抑制。结论：沉默小鼠心肌细胞JP2基因可抑制SK2通道的功能。
Aim:To investigate the relationship between JP 2 and SK2 in the cardiomyocyte .Methods:The recombi-nants of lentiviral vectors targeting JP2 gene were constructed(LV-JP2-RNAi-1,LV-JP2-RNAi-2) for siRNA.The neonatal mouse cardiac myocytes (NMCMs) were infected with the recombinants .The levels of JP2 mRNA and SK2 protein were de-tected by quantitative real-time PCR and Western blot .Results: The JP2 mRNA level of the NMCMs among the four groups were different (F=31.775,P<0.001),which were significantly decreased in LV-JP2-RNAi-1,LV-JP2-RNAi-2 groups compared with control and LV-NC-EGFP groups .Compared with control and LV-NC-EGFP groups , the expression of the SK2 channel protein in the NMCMs with 72 h infection LV-JP2-RNAi-2 was obviously inhibited .Conclusion:Knock-down of JP2 in the mouse cardiomyocyte could suppress the function of SK 2 channel.

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针对雌激素受体(ER)-α36基因的特异性靶序列,构建并鉴定靶向ER-α36基因RNAi慢病毒载体,研究ER-α36沉默后对胃癌细胞增殖的影响。方法:筛选确定的ER-α36基因RNAi有效靶序列,合成靶序列的Oligo DNA,与慢病毒载体(GV307)连接,测序鉴定。转染293T细胞,包装产生慢病毒,感染胃癌SGC7901细胞株。荧光显微镜下观察SGC7901感染后荧光表达情况,real-time PCR和Western blotting方法检测ER-α36的表达变化。用1×10-10mol/L的17β-雌二醇处理沉默ER-α36的SGC7901细胞,用细胞计数法观察细胞增殖能力的变化及检测相关下游信号通路分子Src、ERK1/2、cyclin D1表达的变化。结果:阳性克隆PCR及测序证明成功构建慢病毒载体LV-ER-α36-RNAi,倒置显微镜下观察LV-ER-α36-RNAi慢病毒载体感染率达80%以上。Real-time PCR和Western blotting方法证实四环素(Te T)诱导下LV-ER-α36-RNAi明显抑制SGC7901细胞内ER-α36 mRNA和蛋白质的表达。与对照组相比,沉默ER-α36的SGC7901细胞增殖能力减弱,Src、ERK1/2、cyclin D1蛋白表达明显降低,Src蛋白活化能力减弱(P0.05)。结论:我们构建的Te T诱导靶向ER-α36的vshRNA慢病毒载体LVER-α36-RNAi,可明显沉默ER-α36的表达,为研究ER-α36蛋白
[ ABSTRACT] AIM:To construct a lentiviral vector for stable delivery of the ER-α36 gene and to detect its effect on SGC7901 cell growth.METHODS: The efficient RNAi targeting sequences identified for the ER-α36 gene were screened.The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA.Then it was digested by Xho I and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector.PCR was used to screen the positive clones and sequence.The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line.Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect.17β-estrodial at concentration of 1 ×10 -10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D

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目的：构建靶向犬p53基因慢病毒干扰载体，建立稳定沉默p53基因的WRD/p53-细胞系。方法设计并构建4条针对犬p53基因的特异性shRNA干扰质粒。将构建的慢病毒表达载体（pGMLV-p53）和包装质粒（packaging mix）组成的包装系统共转染293T细胞，包装病毒，收集病毒原液，超滤浓缩，并测定滴度。包装好的慢病毒感染WRD细胞，Western blot和实时荧光定量PCR方法检测不同靶点RNA干扰载体对p53基因的干扰效果，确定有效靶点。针对有效靶点慢病毒颗粒以最适感染复数（multiplicity of infection, MOI）感染WRD细胞，puromycin抗生素筛选稳定感染细胞系WRD/p53-。结果结果显示p53 RNAi慢病毒载体构建成功，成功包装四种不同靶点的p53基因RNA干扰慢病毒,病毒滴度均达到1×109 TU/ml。四种干扰载体慢病毒均具有较好的干扰效果，选用沉默效果最好的pGMLV-p53A1为最优靶点，成功建立p53 RNAi慢病毒载体稳定感染细胞系WRD/p53-。结论成功构建p53 RNAi慢病毒载体，并有效干扰WRD细胞中p53 mRNA和蛋白表达，同时成功筛选并建立p53 RNAi慢病毒稳定感染WRD/p53-细胞系。
Objective To establish a canine cell line with p53 gene knockdown by lentivirus-mediated RNA interference (RNAi). Methods Four pairs of oligonucleotide sequences of p53 gene were synthesized and cloned into the pGMLV-SC5 RNAi vector. Following confirmation by PCR and DNA sequencing, the recombinant pGMLV-p53 plasmids and packaging mix vectors were packaged into mature lentivirus to infect 293T cells. The supernatant of the infected cells was harvested to infect WRD cells, in which p53 expression was detected using Western blotting and real-time PCR. The lentivirus with the strongest p53-silencing effect was packaged for infecting WRD cells at the optimal multiplicity of infection (MOI) to establish a stably infected cell line (WRD/p53-) screened using puromycin. Results The lentivirus carrying p53 shRNA was constructed successfully and a virus titer of 1 × 109 TU/ml. The 4 pGMLV-p53 plasmids all produced strong p53 interference affects, and pGMLV- p53A1 with the strongest ef

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目的构建人Midkine(MK)基因RNA干扰(RNA interference,RNAi)慢病毒载体并鉴定,为其体内外实验研究提供基础。方法设计针对人MK基因序列的4条RNAi靶点序列,分别与慢病毒载体进行连接,获得GV115-MK-1、GV115-MK-2、GV115-MK-3和GV115-MK-4质粒,转染感受态细菌,经PCR及测序技术的鉴定,然后与pHelper 1.0和pHelper 2.0共转染293T细胞,进行慢病毒包装并测定病毒滴度。4种病毒载体感染人乳腺癌MDA-MB-231细胞后,用RT-PCR检测MK mRNA的表达。结果 PCR和测序检测结果证实,插入的MK RNAi核苷酸序列正确,共转染293T细胞后可见大量绿色荧光。浓缩病毒后测定其滴度分别为6×108、5×108、5×108和6×108TU/ml。感染MDA-MB-231细胞后,RT-PCR检测结果表明,GV115-MK-1干扰效果明显,MK基因敲减效率达87.2%(P0.05)。结论成功构建人MK RNAi慢病毒表达载体,并可有效抑制MDA-MB-231细胞MK基因的表达。
Purpose To construct a lentiviral ventor-mediated RNA interference of human midkine ( MK) gene and to provide the ba-sis for further experiment in vivo and in vitro. Methods Four RNAi sequences targeting human MK gene were designed, and cloned into the lentiviral vector to construct lentiviral vectors:GV115-MK-1, GV115-MK-2, GV115-MK-3 and GV115-MK-4. After transfec-tion into competent E. Coli bacteria, the candidate clones were identified by PCR and DNA sequencing. The titer of lentiviruses was determined after 293 T cells were co-transfected with GV115-MK, pHelper 1. 0 and pHelper 2. 0. The four kinds of recombinant lenti-viruses were used to infect human breast cancer cell MDA-MB-231, and the expression levels of MK mRNA were detected by real-time PCR. Results PCR analysis and DNA sequencing confirmed that the four inserted MK RNAi sequences were corrected. Strong green fluorescence was observed in the 293 T cells under the fluorescent microscope after co-transfection. The titer of

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