hERG shRNA 表达载体构建及其稳定转染骨肉瘤细胞系MG-63、 SOSP-9607 的建立

目的:构建hERG钾离子通道蛋白(human ether-a-go-go-related gene potassium channel)shRNA表达载体质粒,获得稳定转染干扰质粒的人骨肉瘤细胞系MG-63、SOSP-9607。方法:将4对合成的寡核苷酸链退火形成双链,连接入pGPU6/GFP/Neo表达载体,并测序鉴定。使用脂质体法将重组的质粒转染至MG-63、SOSP-9607,通过G418筛选建立稳定转染的两种细胞系,采用免疫印迹(Western blot)技术检测hERG蛋白的表达。结果:测序结果证实shRNA与载体连接正确,免疫印迹实验证实hERG蛋白表达显著降低。结论:成功构建了hERG shRNA真核表达载体,获得了稳定表达hERG shRNA的人骨肉瘤细胞系MG-63和SOSP-9607。     

靶向WNT5A基因的shRNA逆转录病毒表达载体的构建与鉴定

目的:构建人WNT5A shRNA逆转录病毒表达载体.方法:根据人WNT5A基因mRNA序列设计并合成两条互补的DNA单链寡核苷酸,将退火后形成的双链连接于pSUPER Retro RNAi质粒,构建WNT5A shRNA逆转录病毒表达载体,经脂质体介导入GPG293细胞,包装成逆转录病毒.用该逆转录病毒感染人鼻咽癌细胞,Western blot法和RT-PCR检测细胞WNT5A的表达.结果:目的序列成功连接于载体并包装成逆转录病毒,免疫印迹检测和RT-PCR检测结果表明构建的WNT5A shRNA逆转录病毒表达载体能显著抑制鼻咽癌细胞WNT5A的表达.结论:成功构建人WNT5A shRNA逆转录病毒表达载体.     

shRNA表达载体构建方法的优化

目的探讨shRNA表达载体的构建方法 ,以加速RNA干扰研究的进程。方法对shRNA表达载体的构建过程进行分析和监测 ,并加以优化。结果发现shRNA表达载体构建的退火过程容易产生障碍 ,经优化退火缓冲液的NaCl含量后 ,能明显提高退火效率及shRNA表达载体构建的成功率。结论shRNA表达载体构建的退火过程需加以关注 ,退火缓冲液中NaCl含量应提高至 2 0 0mmol/L以上为宜     

腺相关病毒介导的RNA干扰抑制HeLa细胞中CTCF的表达

目的:利用RNA干扰(RNAi)稳定抑制HeLa细胞中CTCF的表达。方法:构建能够抑制转录因子CTCF表达的短发夹RNA(shRNA)的腺相关病毒载体,重组病毒感染HeLa细胞后挑取单克隆,用Western印迹法和实时荧光定量PCR检测CTCF的表达水平。结果:获得3株CTCF被显著抑制的HeLa细胞株,Western印迹法和实时荧光定量PCR结果均显示CTCF的表达水平被抑制,其中最明显的被下调76%。结论:shRNA病毒表达载体构建成功,HeLa细胞中CTCF的表达可被长期稳定地抑制。     

生长抑素受体基因特异性shRNA慢病毒表达质粒的构建

目的:设计并合成生长抑素受体SSTR5基因siRNA序列,并构建其短发夹shRNA慢病毒表达质粒.方法:以小鼠SSTR5基因为靶序列,用在线软件分析、设计并合成其有效siRNA,退火形成双链DNA后,与经BamH I和EcoR I双酶切线性化慢病毒表达载体pSHR-Pμro/GFP连接,产生pLV-shSSTR5重组慢病毒质粒.将重组质粒转化大肠杆菌DH 5α感受态细胞,PCR筛选阳性克隆,测序鉴定.结果:构建的重组表达质粒PCR产物为161bp,其中插入的SSTR5-siRNA片段为61bp,测序结果与参考序列完全一致.结论:成功构建了小鼠SSTR5基因特异性shRNA慢病毒表达质粒,为进一步采用RNAi技术研究小鼠SSTR5基因表达对其生长情况的影响奠定了基础.     

靶向FTL基因的shRNA真核表达载体的构建及鉴定

目的:为了构建铁蛋白轻链(ferritin light chain,FTL)的短发夹小干扰RNA表达载体,并提供进一步研究FTL功能的平台。方法:针对小鼠FTL基因的特异性位点设计并合成特异性长度约65bp的短发夹状小干涉RNA(shRNA)寡核苷酸序列,退火形成双链后将其插入真核表达载体pRNA-U6.1/neo的限制性内切酶HindⅢ和Bam HⅠ之间中,构建成FTL基因的干扰载体(FTL shRNA)。经测序鉴定正确后,用lipofectamineTM2000将FTL shRNA及其对照空载体pRNA-U6.1/neo分别转染到小鼠腹腔单核巨噬细胞(RAW264.7)中,提取蛋白后利用Western blot技术检测细胞中FTL蛋白的表达。结果:得到与预期目的片段大小相似的重组载体,测序结果显示所构建的小鼠FTL shRNA质粒序列正确。细胞内的FTL表达被FTL shRNA成功干扰,效率高达80%。结论:实验成功构建了小鼠FTL基因的短发夹小RNA干扰表达载体FTL shRNA。     

人线粒体转录终止因子3基因RNA干扰表达载体的构建筛选与干扰效果评价

目的:构建人线粒体转录终止因子3(MTERF3)基因的短发夹RNA(shRNA)干扰表达载体,并在mRNA和蛋白质水平对其干扰效率进行验证,以检测和筛选出干扰效率最优的shRNA表达载体。方法:根据人MTERF3基因全长cDNA序列,利用Oligoengine在线软件设计4个shRNA序列,合成4条互补寡核苷酸链,退火成双链后克隆至psiU6.1载体,得到4个重组干扰质粒psi-MTERF3-1～psi-MTERF3-4,并通过PCR和DNA测序对其进行鉴定;将重组干扰质粒利用脂质体介导瞬时转染He La细胞,转染48 h后采用real time RT-PCR检测4个干扰质粒对MTERF3 mRNA表达水平的影响,采用Western印迹检测其对MTERF3蛋白表达水平的情况,并筛选出最有效的shRNA干扰质粒。结果:PCR鉴定和DNA测序鉴定证实重组质粒中已插入目的DNA序列;real time RT-PCR和Western印迹结果表明4个重组载体均可以显著降低人MTERF3基因mRNA和蛋白质的表达水平(P0.05),其中以pSi-MTERF3-4的干扰效率最优,对MTERF3 mRNA表达的抑制率为70.1%,对MTERF3蛋白表达的抑制率为72.2%。结论:在人宫颈癌He La细胞系筛选出有效干扰MTERF3基因表达的shRNA序列,构建了针对MTERF3基因的RNA干扰真核表达载体且能够有效抑制目的基因的表达,为进一步研究人MTERF3在宫颈癌发生发展中的作用机制提供了实验基础。     

绿色荧光蛋白标记的TGF-β1shRNA真核表达载体的构建及鉴定

目的：构建小鼠转化生长因子β1（TGF-β1）短发夹RNA（shRNA）真核表达载体,探讨TGF-β1在血管发育中的调控作用。方法：根据GenBank小鼠TGF-β1mRNA序列,设计合成三对短链寡核苷酸,退火后形成双链DNA并克隆至入门载体DEN_mH1c。将插入目的基因片段的入门载体与带有绿色荧光蛋白（GFP）标签的shRNA真核表达载体pDS_hpEy进行LR重组反应,完成三个TGF-β1shRNA表达载体的构建,分别命名为pDS_Ta,pDS_Tb和pDS_Tc。经测序确认后,转染小鼠成纤维细胞（NIH／3T3）,筛选稳定表达的细胞克隆,以RT-PCR及Westem blot方法检测转染后TGF-β1mRNA和蛋白表达。结果：RT-PCR和Western blot显示pDS_Tc可明显下调NIH／313细胞TGF-β1的mRNA和蛋白表达,mRNA下调约为70％,蛋白表达减少约65％。结论：GFP标签TGF-β1shRNA表达载体能够阻断TGF-β1基因表达,可作为研究TGF-β1调控血管发育机制的一个工具,为阐明TGF-β1信号传导通路奠定基础。     

端粒酶shRNA 慢病毒载体的构建及转染人类胚胎干细胞

目的:构建端粒酶shRNA慢病毒载体及建立端粒酶稳定干扰的人类胚胎干细胞系。方法:将端粒酶基因特异性shRNA靶序列与慢病毒载体PLKO.1-puro连接、转化、挑取阳性克隆进行PCR及测序鉴定;利用包装细胞293T获得重组的慢病毒,感染人类胚胎干细胞,分为干扰组ShTert、载体组vector和野生型组wt;Realtime-PCR检测端粒酶mRNA的表达。结果:经PCR和DNA测序鉴定,成功构建端粒酶特异性shRNA慢病毒载体,并感染人类胚胎干细胞;经检测shTert组端粒酶mRNA表达较vector和wt组明显降低,vector组和wt组之间无明显差异。结论:通过成功构建的端粒酶特异性shRNA慢病毒载体对人类胚胎干细胞的转染实现了对其端粒酶mRNA的调控。     

体外RNA干扰下调DEPTOR表达对人多发性骨髓瘤细胞增殖和凋亡能力的影响

目的:研究探讨应用RNA干扰技术构建人DEPTOR基因的shRNA慢病毒载体,鉴定在多发性骨髓瘤RPMI-8226细胞上的沉默效果,并评价DEPTOR对人多发性骨髓瘤细胞增殖和凋亡能力的影响.方法:设计4个DEPTOR siRNA序列,化学合成后退火形成双链,克隆到酶切的hU6-MCS-CMV-EGFP(GV115)-shRNA慢病毒载体.重组质粒经PCR测序鉴定后,脂质体介导下转染293T细胞,Western blot检测DEPTOR的表达,筛选出干扰效果最佳的GV115-shRNA.将筛选的GV 115-shRNA与慢病毒包装质粒共转染293T细胞生成病毒,收集病毒上清并浓缩,测定浓度.将病毒颗粒感染多发性骨髓瘤RPMI-8226细胞,应用Real-time PCR方法从mRNA水平及Western blot方法从蛋白水平检测DEPTOR的沉默效果.运用四甲基偶氮唑蓝(MTT)法检测骨髓瘤细胞增殖能力的变化;流式细胞仪检测细胞凋亡;用Western blot分析cleaved caspase-3和cleaved PARP的变化.结果:构建的慢病毒载体shRNA的PCR鉴定和测序正确,成功筛选出最有效抑制DEPTOR表达的shRNA2序列,包装病毒后滴度达到1×109TU/ml.慢病毒感染RPMI-8226细胞后可表达EGFP,Real-time PCR和Western blot检测DEPTOR的表达明显下降.下调DEPTOR基因可显著减慢骨髓瘤细胞的生长速度(P＜0.05),肿瘤细胞的凋亡率上升(P＜0.01).DEPTOR shRNA上调cleaved caspase-3、cleaved PARP和Bax表达,下调Bcl-2及PI3K/Akt信号通路的表达(P＜0.01).结论:成功构建了DEPTOR shRNA重组慢病毒载体,其转染多发性骨髓瘤RPMI-8226细胞后显著抑制了DEPTOR的表达.DEPTOR shRNA可以有效地诱导骨髓瘤细胞的凋亡,并抑制肿瘤细胞的增殖.PI3K/Akt信号通路可能参与了其凋亡过程.     

Alternative methods for the efficient construction of short hairpin RNA expression vectors

Short hairpin RNA (shRNA)-mediated RNA interference has become a basic technique in modern molecular biology and biochemistry for studying gene function and biological pathways. Here, we report two alternative and efficient methods to construct shRNA expression vectors based respectively on multiple-step sequential PCR and primer extension–homologous recombination (PE-HR). Neither method requires synthesizing long oligonucleotides containing hairpin sequences as used in traditional approaches. The hairpin sequences may produce mutations during oligo synthesis, pose problems in annealing, and lead to inefficient cloning. The PE-HR method further provides rapid and economical construction of shRNA expression vectors without needing the ligation procedure.     

An advanced blue-white screening method for construction of shRNA expression vectors

Short hairpin RNA (shRNA) encoded within an expression vector is an effective tool for exploration of gene function in mammalian cells. Many of the current methods for constructing shRNA expression vectors require cumbersome and time-consuming procedures for identification of the desired recombinants. We have developed a highly efficient and less labor-intensive cloning method that allows the construction of shRNA expression vectors in one day and with minimal effort. This advanced blue-white screening technique was developed by combining the reconstitution of ideal lacO with TA cloning. The DNAs are simply ligated into the destination vectors and, following transformation, a desired recombinant event will give a typical blue colony. In addition, we have used this cloning method for the construction of targeting reporter expression vectors to measure the efficacy of the corresponding shRNA. We constructed 122 functional shRNA expression vectors and sequencing of the positive cloning vectors confirmed a high degree of accuracy. Only three short DNA primers are needed for constructing both shRNA and targeting reporter expression vectors. This advanced blue-white screening system is a powerful tool for the high-throughput assay of RNAi libraries.     

The comparative and functional study between two construction methods of shRNA expression vector targeted <Emphasis Type="Italic">LMP1</Emphasis> gene encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.      

The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.     

The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells,we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis,RT-PCR and western blot.pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method.Furthermore,the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors.According to our research,we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells,and also provides a novel application of RNA interference technology against-EBV.     

猪繁殖与呼吸综合征病毒ORF2～4特异siRNA的筛选及其抑制病毒复制效果的研究

RNA干扰(RNAi)技术是基因功能研究的有效工具,为了了解猪繁殖与呼吸综合征病毒(PRRSV)次要结构蛋白GP2、GP3、GP4在病毒复制中的作用,针对各自的编码基因ORF2、ORF3、ORF4分别选取4个小干扰RNA(siRNA)位点(共12个),构建相应的短发夹RNA(shRNA)表达载体,转染MARC-145细胞后,通过荧光定量PCR和病毒滴度检测干扰效果。筛选了可以减少GP2、GP3、GP4相应基因mRNA含量的ORF2、ORF3、ORF4特异shRNA表达载体,病毒效价滴定表明shRNA表达载体处理细胞可以减少GP2、GP3、GP4相应基因mRNA含量,细胞培养上清中的病毒滴度比对照低184~4.65倍。     

An Infinitely Expandable Cloning Strategy plus Repeat-Proof PCR for Working with Multiple shRNA

Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios.     

A simple and economical short-oligonucleotide-based approach to shRNA generation

RNAi (RNA interference) has become a popular means of knocking down a specific gene in vivo. The most common approach involves the use of chemically synthesized short interfering RNAs (siRNAs), which are relatively easy and fast to use, but which are costly and have only transient effects. These limitations can be overcome by using short hairpin RNA (shRNA) expression vectors. However, current methods of generating shRNA expression vectors require either the synthesis of long (50-70 nt) costly oligonucleotides or multi-step processes. To overcome this drawback, we have developed a one-step short-oligonucleotides- based method with preparation costs of only 15% of those of the conventional methods used to obtain essentially the same DNA fragment encoding shRNA. Sequences containing 19 bases homologous to target genes were synthesized as 17- and 31-nt DNA oligonucleotides and used to construct shRNA expression vectors. Using these plasmids, we were able to effectively silence target genes. Because our method relies on the one-step ligation of short oligonucleotides, it is simple, less error-prone, and economical.     

High-throughput isolation of ultra-pure plasmid DNA by a robotic system

Background  

Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %).