组织特异RNAi转基因小鼠模型的构建

RNAi是一种行之有效的基因沉默的新方法,被广泛地应用于基因功能的研究、疾病的治疗以及新型疫苗的研制等领域.本研究通过原核显微注射干扰载体的方法制备转基因小鼠.选用皮肤组织特异表达的人源角蛋白14（K14）基因启动子（2000bp）作为表达载体启动子,成功地驱动融合表达载体EGFP-shRNA进行干扰片段前体的转录,进而生成成熟的干扰片段,靶向小鼠BMP4基因使其发生沉默.所得到的转基因小鼠及其杂交后代经PCR和Southern杂交鉴定,结果表明外源基因准确无误地整合到小鼠基因组.Northern杂交结果证明,小干扰RNA在皮肤组织中有较高水平的表达,在肺和肠组织中有较低水平的表达.研究结果表明,利用PolⅡ型（K14）启动子驱动shRNA融合转录本的表达,在特定组织高表达siRNA,从而达到抑制特定组织目的基因表达的技术路线是可行的.同时为利用K14启动子进行毛囊相关基因干扰研究积累了基础数据,为制备组织特异抑制基因表达的转基因大家畜提供了一个参考方法.     

发夹RNA(shRNA)在哺乳动物RNAi研究中的应用

在哺乳动物的RNAi研究中,载体表达的shRNA分子比细胞同时表达的siRNA分子的正义链与反义链对靶基因的抑制效率要高。shRNA可由PolⅢ的启动子在体内表达产生,酶切cDNA和shRNA芯片是产生shRNA的最新方法。对shRNA的设计应注意靶基因序列、环序列以及载体酶切位点的选择。诱导表达shRNA的载体系统的表达效率有所差异,质粒载体转染效率尚不稳定,且持续时间短,通过病毒载体介导是目前进行基因敲除最有效的工具。     

非完全互补shRNA对RNA聚合酶II启动子调控的RNAi的影响

RNA干扰（RNAi）是一种转录后基因沉默技术,可有效诱导序列特异性基因沉默．由RNA聚合酶Ⅱ启动子调控表达的小发卡RNA可有效介导RNAi效应,为组织特异性基因沉默提供了一条新的途径．但是,由RNA聚合酶Ⅱ启动子调控表达的小发卡RNA（shRNA）在序列上与靶基因非完全互补对RNAi效应的影响鲜有报道．本文初步探索RNA聚合酶Ⅱ启动子调控表达的shRNA碱基发生突变或缺失对RNAi效应的影响．研究表明,靶向hTERT mRNA的碱基突变shRNA显著降低RNAi效应,而靶向GFP mRNA的碱基缺失shRNA对RNAi效应没有显著影响．本研究为非完全互补shRNA对RNAi效应的进一步深入研究提供了理论与实验依据．     

siRNA抑制HIV-1基因表达的研究

RNA干扰(RNAinterfering,RNAi)现象是动植物体内的一种序列特异性的、转录后基因沉默的过程.在哺乳动物细胞中,19～25个核苷酸长短的双链siRNA(smallinterferingRNA)可有效地抑制基因表达.利用pBS/H1SP载体,它表达的双链RNA的转录产物可以用来抑制特异性HIV-1基因的表达.在siRNA实验中,为了比较由H1启动子转录的siRNA在细胞内的作用,使用以绿色荧光蛋白(EGFP)为报告基因的载体——pEGFP-C1质粒,在荧光显微镜下,很容易地看到EGFP在细胞中表达.将HIV-1siRNA表达载体与表达相应EGFP-HIV融合基因的质粒,共转染人胚肾293细胞,结果表明,和对照质控载体相比,转染了pHIV-siRNA质粒的细胞中EGFP-HIV的表达得到显著抑制.通过该途径,筛选出能抑制HIV-1基因表达的有效siRNA.此外,还在同一载体上表达两种或三种siRNA,分别针对不同的HIV基因,获得了良好的抑制效果.     

沉默FNBP1基因对人HL-7702细胞体外增殖及迁移能力的影响

为了研究沉默FNBP1(formin-binding protem1)基因对细胞体外增殖以及对细胞迁移能力的影响,设计并构建了3个靶向FNBP1 siRNA干扰载体(Si-1,Si-2,Si-3),经酶切和DNA测序鉴定后转染人正常的肝细胞HL-7702。RT-PCR和Western blot检测瞬时转染细胞中FNBP1基因的干扰效率,筛选出特异高效的RNAi靶点。并用G418筛选稳定表达了RNAi的细胞株,用XTT法检测细胞体外增殖率,划痕法检测细胞的迁移能力。结果显示:酶切和测序结果证明干扰质粒构建正确;转染特异性的干扰质粒(Si-1、Si-2、Si-3)细胞的FNBP1基因表达量与对照组相比均有所降低,在蛋白质水平表达也明显受到抑制,且重组干扰质粒Si-2的干扰效应较强。XTT检测结果表明,构建的沉默FNBP1重组质粒Si-2能显著抑制HL-7702细胞的体外增殖能力,但并不直接影响HL-7702细胞的迁移能力。     

维甲酸受体β基因RNAi表达质粒的构建及其对A549细胞增殖和分化的影响

目的：利用RNA干扰（RNAi）技术,构建针对维甲酸受体（RAR）β基因的小干扰RNA（siRNA）表达质粒,诱导RARβ基因沉默,并观察其对肺癌细胞A549株的细胞周期和增殖的影响。方法：依据设计siRNA的原则。针对人RARβ的mRNA序列,设计并合成编码siRNA的2条寡核苷酸序列,经退火成互补双链,再克隆到pSUPER-NEO-GFP真核表达载体中构建重组体pSUPER-RAR[β转染至A549细胞中,以空质粒和RARβ高表达质粒转染为对照,用Western印迹检测RARβ基因的表达,并采用M1Tr试验检测转染后细胞株的增殖和细胞分化情况。结果：表达人类RARβ基因的siRNA重组表达质粒构建成功;MTT试验结果表明,转染的A549-RARβ-si细胞增殖能力降低。结论：采用RNAi技术特异阻断RARB基因表达,通过转染A549细胞,可使其细胞形态发生变化,并抑制其细胞生长。     

人PINK1-shRNA载体的构建及对神经母细胞瘤细胞线粒体的影响

摘要 目的：构建筛选人源靶向特异PINK1-shRNA敲减质粒,转染神经母细胞瘤SH-SY5Y细胞并验证该质粒转染后对PINK1基因的敲减效率,观察对细胞线粒体形态的影响。方法：构建两对人源PINK1-shRNA序列(编号分别为PINK1-shRNA-39和PINK1-shRNA-42),将这2对干扰序列连接在载体上形成重组载体,经测序验证后,将空载体和两对敲减质粒分别转染SH-SY5Y细胞,获得基因敲减的细胞模型,用CCK-8法检测细胞的存活率,用荧光定量PCR方法确定PINK1基因的敲减效率,用蛋白质免疫印迹法验证细胞内PINK1的表达水平是否发生改变,用激光共聚焦显微镜观察线粒体的形态是否发生了改变。结果：我们提取的质粒,经测序结果显示,质粒载体构建成功;转染细胞后,CCK-8 法检测细胞存活率发生了降低,与正常组比较,PINK1-shRNA-39和PINK1-shRNA-42敲减质粒组,细胞的存活率分别降低了13.7%（P＜0.05）和14.1%（P＜0.05）;荧光定量PCR结果显示,与正常组相比,PINK1-shRNA-39组和PINK1-shRNA-42组敲减质粒转染的细胞内PINK1基因的表达分别降低了24.1%（P＜0.01）和36.7%（P＜0.01）;蛋白质印迹法结果显示,与正常组相比,两对质粒分别转染细胞后,PINK1-shRNA-39和PINK1-shRNA-42敲减质粒转染的细胞内PINK1蛋白的表达水平降低,差异有统计学意义(P＜0.01),PINK1-shRNA-42敲减质粒转染的细胞内PINK1蛋白的表达水平有效降低更明显;与正常组比较,激光共聚焦显微镜观察到基因敲减两组细胞的线粒体部分发生断裂,碎片较多,基因敲减两组的线粒体的形态因子显著降低,差异有统计学意义（P＜ 0.01）。结论：成功构建了人源的PINK1基因敲减的质粒,并将敲减的质粒成功转染至SH-SY5Y 细胞中,细胞内PINK1基因的mRNA和蛋白的表达水平降低,且细胞线粒体的形态发生了改变。     

Bcl-2/C-Raf双基因“敲低”抑制人结肠癌细胞增殖

 RNA干扰是一种具有序列特异性的基因沉默,能够触发具有相应序列的mRNA的降解.构建具有双靶点的RNAi质粒表达载体,与单靶点表达载体比较,探讨其对结肠癌细胞增殖的抑 制作用.本研究分别构建了针对Bcl-2、C-Raf 和Bcl-2/C-Raf靶基因的质粒表达载体,通过Lipofectamine TM2000介导转染人结肠癌细胞系HCT-8后,检测相应转染组靶基因的mRNA和蛋白质表达量,测定各组细胞活性,研究RNAi对各组癌细胞增殖的抑制率.结果表明,分别转染3种质粒表达载体后,3组结肠癌细胞中相应靶基因的mRNA和蛋白质表达量均降低;转染双靶点干扰质粒的试验组;其细胞活性低于单靶点组;对于针对Bcl-2, C-Raf和Bcl-2/C-Raf基因的3组干扰实验,RNAi对结肠癌细胞增殖的抑制率分别为43.87%,40.64% 和63.85%.RNAi是结肠癌细胞中的一种功能途径,以质粒作为表达载体,同时具备Bcl-2/C-Raf双靶点的表达载体,对结肠癌细胞增殖的抑制作用要明显优于单靶点表达载体,双靶点质粒表达载体在结肠癌的基因治疗中是有潜力的.     

鸡源细胞基因沉默及快速筛选的实用型双标记RNAi载体

利用人H1 RNA启动子、EGFP基因及Neomycin抗性基因,构建用于禽类细胞基因持续沉默和快速筛选的实用型RNAi载体。在将pCDNA3.1(+)载体上的SV40启动子替换为鸡源的β-actin启动子后,装入EGFP基因表达框以及用于驱动外源shRNA转录的人H1 RNA启动子,构建成同时具有EGFP和Neomycin抗性双标记的RNAi载体,并为载体引入独特设计的含媒介序列的多克隆位点以方便外源shRNA编码小片断插入后的快速筛选,载体设计非常实用。插入靶向EGFP和sIgM λ基因的shRNA编码序列后分别瞬时转染DF-1和DT40细胞,结果显示靶基因表达得到了明显抑制。联用EGFP和Neomycin双标记快速筛选sIgM λ轻链基因稳定沉默的DT40细胞克隆的结果也证实,H1启动子转录shRNA的干扰效果是高效的,双标记筛选策略不仅有效而且方便、快捷。     

RNA干扰NUP88基因对人乳腺癌MCF-7细胞生长及侵袭力的影响

目的： 构建重组慢病毒介导的NUP88-shRNA载体,通过RNAi技术分别观察沉默NUP88后对MCF-7增殖,粘附,侵袭和转移情况的影响,为乳腺癌的临床基因治疗寻找新的靶点。方法： 构建NUP88重组慢病毒表达载体,包装后检测滴度,以最佳复感染指数转染乳腺癌MCF-7细胞,利用RT-PCR和Western blot检测各组MCF-7细胞中mRNA和蛋白的表达效率;MTT法和流式细胞仪检测法,检测NUP88基因被干扰后对MCF-7细胞增殖和凋亡的影响;细胞侵袭实验检测NUP88基因被干扰后对MCF-7侵袭力的影响。结果 四组病毒及一组阴性对照均构建成功,滴度均为4E+8TU/ml;RT-PCR和Western blot检测,结果表明：经NUP88-shRNA转染的MCF-7细胞组NUP88 mRNA和蛋白质的表达与经阴性转染组和空白MCF-7细胞组相比,差异明显具有统计学意义（P<0.01）;测定NUP88-shRNA1组沉默效率最高,沉默率可达到86%;MTT法结果表明：实验组经NUP88-shRNA1慢病毒转染后细胞增殖程度显著减少,与空白组和对照组相比有显著性差异（P<0.05）。流式细胞仪检测三组MCF-7细胞凋亡结果表明：实验组经慢病毒转染后细胞凋亡率显著增加,与对照组和空白组相比有显著性差异（P<0.05）;细胞侵袭实验表明：在肿瘤细胞常规培养24h后,实验组与空白组和阴性对照组比较,穿膜细胞数量明显减少,具有显著性差异（P<0.05） 结论： NUP88重组慢病毒可以通过RNAi成功抑制MCF-7中NUP88基因的表达,并能显著抑制其增殖及远处的侵袭能力。     

Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology

Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II (Pol II)-directed promoter in a fusion form can disrupt the targeted gene expression. Here, we report that using both luciferase and antiapoptotic survivin genes as targets, both siRNA and shRNA approaches significantly silenced the targeted gene expression in cancer cells. We further demonstrated that shRNAs transcribed from an RNA Pol II-mediated promoter in a green fluorescent protein (GFP) fusion form at the 3'-untranslated region silenced luciferase and survivin expression as well, suggesting that the extra RNA sequence outside of the shRNA hairpin does not disrupt shRNA function. We also showed that silencing of survivin expression selectively induces apoptosis in transfected cells. Together, we have validated multiple approaches of RNAi technology using both survivin and luciferase genes as targets and demonstrated for the first time that GFP-shRNAs transcribed from an RNA Pol II-mediated promoter could mediate gene silencing, which may lead to new directions for the application of RNAi technology.     

An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi

RNA interference (RNAi) mediates gene silencing in many eukaryotes and has been widely used to investigate gene functions. A common method to induce sustained RNAi is introducing plasmids that synthesize short hairpin RNAs (shRNAs) using Pol III promoters. While these promoters synthesize shRNAs and elicit RNAi efficiently, they lack cell specificity. Monitoring shRNA expression levels in individual cells by Pol III promoters is also difficult. An alternative way to deliver RNAi is to use Pol II-directed synthesis of shRNA. Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy. Here we demonstrate a new Pol II system that directs efficient shRNA synthesis and mediates strong RNAi at levels that are comparable with the commonly used Pol III systems. In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression. This system may be adapted for in vivo shRNA expression and gene silencing.     

Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.     

Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)-expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)-the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.     

Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference

RNA interference (RNAi) mediated by short interfering RNA (siRNA) is a powerful reverse genetics tool and holds enormous therapeutic potential for various diseases, including parasite infections. siRNAs bind their complementary mRNA and lead to degradation of their specific mRNA targets. RNAi has been widely used for functional analysis of specific genes in various cells and organisms. In this paper, we tested the potential of silencing the expression of the Mago nashi gene in Schistosoma japonicum by siRNAs derived from shRNA expressed by mammalian Pol III promoter H1. Schistosomula, transformed from cercariae by mechanical shearing of the tails, were electroporated with Mago nashi shRNA expression vector. Aliquots of parasites were harvested at days 1, 3, and 5 after electroporation, respectively. Levels of Mago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis. The results showed that shRNA expressed from mammalian Pol III promoter H1 specifically reduced the levels of Mago nashi mRNA and proteins in S. japonicum. Changes in testicular lobes were apparent when parasites were introduced into mammalian hosts. Thus, vector-mediated gene silencing is applicable to S. japonicum, which provides a means for the functional analysis of genes in this organism.     

Pol II–Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.     

RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis

The RNA interference technique is a powerful tool to understand gene function. Intriguingly, RNA interference cannot only be used for cells in vitro, but also in living organisms. Here, we have adapted the method for use in the chick embryo. However, this technique is limited by the uncertainty in predicting the RNAi transfection efficiency and site in the embryo. Hence, we elaborated a modified vector system, pEGFP-shRNA, which can coexpress enhanced green fluorescent protein (EGFP) and short hairpin RNA (shRNA) simultaneously to facilitate analysis of gene silencing in chicken embryos. We tested the silencing of two highly conserved genes (cAxin2, cParaxis), which play crucial roles in chicken embryonic developmental processes. For each target gene, four to five small DNA inserts, each of them encoding one shRNA, were selected and cloned individually to the vector downstream of the Pol III promoter (either human H1 or U6 promoter), which shared with highly conserved motifs in human and chicken. The pEGFP-shRNA constructs were electroporated into the neural tube or somites. After subsequent re-incubation of 24 h, the EGFP expression, with green fluorescent signal, indicated the transfected regions in the neural tube or somites. The EGFP expressing embryos were further submitted into the process of in situ hybridization for examination of the silencing effects. The results show that the EGFP signal in transfected areas correlated with the silencing of the target genes (cAxin2, cParaxis). The cAxin2 expression was inhibited by shRNAs of either targeting the RGS domain or the DAX domain coding region. The cParaxis mRNA level in transgenic somites and the related migratory myogenic population was also reduced. The results suggest that our novel dual expression EGFP-shRNA system opens a new possibility to study gene function in a convenient and efficient way.     

猪Nanog基因四环素诱导干扰载体的构建和鉴定

Nanog基因是在早期胚胎和干细胞等多能性细胞中特异表达的重要基因,但有关猪Nanog基因功能的相关研究甚少。四环素诱导干扰载体是一种可通过四环素等药物条件性诱导干扰目的基因的载体,尤其适用于在发育过程中起着关键作用的基因沉默。常规的四环素干扰系统为二元载体,与一元载体相比获得针对特定基因干扰的稳定细胞系所需周期更长。首先通过构建pGenesil 1.0-shRNA重组干扰载体,瞬时转染稳定过表达猪Nanog基因的猪胎儿成纤维细胞后通过Realtime-PCR筛选出干扰效率可达80%以上的干扰片段。之后将筛选得到的干扰片段插入到改造的一元四环素诱导干扰载体TREsilencer,对稳定表达猪Nanog基因的猪胎儿成纤维细胞进行了瞬时转染。实验分别通过光密度检测以及Realtime-PCR检测了不同浓度doxycycline的诱导效率和干扰效率。结果表明,所构建的四环素诱导干扰载体TREsilencer-shRNA5随着四环素浓度的增加,诱导Nanog基因的干扰效率增加,在处理浓度为1μg/ml时干扰效率可达70%以上,为后续得到可诱导的稳定干扰猪Nanog基因的细胞系和进一步研究猪Nanog基因功能奠定了基础。     

An enhanced U6 promoter for synthesis of short hairpin RNA

Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo and, therefore, may be developed to treat diseases caused by dominant, gain-of-function type of gene mutations. These diseases develop in people bearing one mutant and one wild-type gene allele. While the mutant is toxic, the wild-type performs important functions. Thus, the ideal therapy must selectively silence the mutant but maintain the wild-type expression. To achieve this goal, we designed an shRNA that selectively silenced a mutant Cu,Zn superoxide dismutase (SOD1^G93A) allele that causes amyotrophic lateral sclerosis. However, the efficacy of this shRNA was relatively modest. Since the allele-specific shRNA has to target the mutation site, we could not scan other regions of SOD1 mRNA to find the best silencer. To overcome this problem, we sought to increase the dose of this shRNA by enhancing the Pol III promoter. Here we demonstrate that the enhancer from the cytomegalovirus immediate-early promoter can enhance the U6 promoter activity, the synthesis of shRNA and the efficacy of RNA interference (RNAi). Thus, this enhanced U6 promoter is useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region.