Development of strategies for conditional RNA interference

BACKGROUND: RNA interference (RNAi) represents a powerful tool with which to undertake sequence-dependent suppression of gene expression. Synthesized double-stranded RNA (dsRNA) or dsRNA generated endogenously from plasmid or viral vectors can be used for RNAi. For the latter, polymerase III promoters which drive ubiquitous expression in all tissues have typically been adopted. Given that dsRNA molecules must contain few 5' and 3' over-hanging bases to maintain potency, employing polymerase II promoters to drive tissue-specific expression of RNAi may be problematic due to potential inclusion of nucleotides 5' and 3' of siRNA sequences. METHODS: To circumvent this, polymerase II promoters in combination with cis-acting hammerhead ribozymes and short-hairpin RNA sequences have been explored as a means to generate potent dsRNA molecules in tissues defined by the promoter in use. RESULTS: The novel constructs evaluated in this study produced functional siRNA which suppressed the enhanced green fluorescent protein (eGFP) both in vitro and in vivo (in mice). Additionally, the constructs did not appear to elicit a significant type-1 interferon response compared to traditional H1-transcribed shRNA. CONCLUSIONS: Given the potential 'off-target' effects of dsRNAs, it would be preferable in many cases to limit expression of dsRNA to the tissue of interest and moreover would significantly augment the resolution of RNAi technologies. Notably, the system under evaluation in this study could readily be adapted to achieve this objective.     

A Novel Method to Convert a DNA Fragment Inserted into a Plasmid to an Inverted Repeat Structure

Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.     

The histone deacetylase inhibitor sodium butyrate inhibits baculovirus-mediated transgene expression in Sf9 cells

Recent studies have indicated that histone deacetylase inhibitors (HDACis) could enhance and prolong expression of exogenous genes delivered by various viral vehicles in mammalian cells, including baculovirus vectors. In this study, the effects of HDACis on expression of a baculovirus-mediated eGFP reporter gene under control of baculovirus late promoter p10 in Sf9 cells were evaluated. It was found that sodium butyrate (NaBu) decreased the expression level of the target gene driven by p10 promoter by four to fivefold. Moreover, addition of NaBu increased DNaseI-sensitivity of transgene p10 promoter region and did not influence viral DNA replication. FACS assay has shown that both NaBu and fluorodeoxyuridine (FdUrd) blocked Sf9 cells at G1 phase and inhibited the target gene expression. Another HDACi, trichostatin, had little effects on both cell cycle and Ac-p10-eGFP expression, strongly suggesting that cell cycle arrest accounts for the mechanisms by which NaBu inhibits Ac-p10-eGFP expression. The inhibiting effects of NaBu on baculovirus transgene expression in Sf9 cells are promoter specific since the enhancement of NaBu on transgene expression in insect and mammalian cells are mediated by baculovirus harboring a murine cytomegalovirus (mCMV) immediate early promoter. This study was aimed at improving the productivity of the recombinant proteins and providing a better understanding of the epigenetic regulation of baculovirus gene expression.     

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.     

Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer

Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×10⁹ Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.     

短发夹 RNA 介导 RNA 干扰的时间 和剂量效应研究

用 RNA 干扰 (RNA interference , RNAi) 技术抑制哺乳动物细胞中外源报告基因的表达,以探讨该过程中 RNAi 作用的剂量和时间效应 . 应用 Lipofectamine 2000 将外源报告基因的表达载体与编码短发夹 RNA (short hairpin RNA , shRNA) 的质粒共转染 HEK293H 细胞,观察 shRNA 载体对报告基因的抑制效应 . 转染后, shRNAs 的瞬时表达可特异地抑制细胞内报告基因的表达 . 在共转染后 12 , 24 , 48 , 60 , 72 , 96 h 时检测 EGFP (enhanced green fluorescent protein , EGFP) 基因 mRNA 及蛋白质表达水平,结果显示, EGFP mRNA 及蛋白质表达在 12 h 时略有降低, 24~48 h 时表达逐渐降低, 48~72 h 时降低最明显,其后 EGFP 表达水平逐渐恢复 . 提示该过程中 RNAi 效应呈现由弱到强、又由强到弱的逐渐消逝趋势 . 共转染一系列剂量比例的 EGFP 干扰载体与靶载体的结果表明,在一定剂量范围内, RNA 干扰载体所介导的抑制效应与干扰载体剂量大小有关,当其剂量进一步加大足以抑制外源基因表达时,抑制效应则维持在一“平台期” . 此外,通过 RNAi 抑制 HeLa 细胞、 HEK293 细胞中荧光素酶基因的表达, 荧光素酶活性变化也表现出上述类似的效应 . 这些结果表明,在体外哺乳动物细胞中,基于表达载体的 RNAi 作用呈现剂量和时间依赖性效应 . 这为基于载体表达的 RNAi 技术应用研究提供了一定的理论参考及依据 .     

Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.     

RNA干扰对MDA-MB-231细胞中CaSR基因表达的影响

目的:运用RNA干扰技术,观察siRNA表达载体在乳腺癌细胞MDA-MB-231中对CaSR基因表达的影响。方法:构建靶向CaSR基因的RNA干扰表达载体,用脂质体转染乳腺癌细胞株MDA-MB-231,运用Real-Time荧光定量PCR,Westernblot技术分别从mRNA以及蛋白表达水平检测CaSR基因表达的变化。结果:所构建的质粒载体成功的在MDA-MB-231细胞中抑制了CaSR mRNA及其蛋白的表达。与对照组相比,psiRNA-CaSR载体对CaSR mRNA的抑制率达到65%,对CaSR蛋白抑制率约为70%。结论:实验证明所设计的shRNA片段可以有效地抑制CaSR基因的表达,为下一步研究工作奠定了基础。     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.     

汉坦病毒H8205株G1P基因片段重组杆状病毒表达载体的构建及表达

将汉坦病毒H8205株G1P基因的保守序列(约1000bp)作为目的基因插入到BactoBac杆状病毒表达系统的pFastBacHTb供体质粒中,利用Tn7转座子同BacmidDNA同源重组,获得了含目的基因片段的重组杆状病毒DNA,并利用其转染Sf9昆虫细胞,72h后收集细胞悬液,再用该悬液侵染Sf9昆虫细胞,48h后收获病毒.采用IFA分析收获的产物,观察到了特异性的荧光,并且采用SDSPAGE和Western印迹也获得了与预期一致的结果.证明感染后的Sf9昆虫细胞所表达的蛋白中含有能与抗汉坦病毒H8205株多克隆抗体特异性结合目的蛋白.研究表明,采用杆状病毒表达系统可以成功表达出汉坦病毒H8205株包膜糖蛋白G1基因片段,为开发适合的以G1P为抗原的汉坦病毒诊断试剂进行了前期的探索.     

RNA interference‐mediated knockdown of IAP in Lygus lineolaris induces mortality in adult and pre‐adult life stages

In recent years, RNA interference (RNAi) has been validated as a viable approach for functional genetic studies in non‐model organisms. In this report we demonstrate the efficacy of RNAi in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). A L. lineolaris inhibitor of apoptosis gene (LlIAP) has been identified and cloned. The translated sequence encodes a 381 amino acid protein similar to other insect IAPs and contains two conserved baculovirus inhibitor of apoptosis protein repeat (BIR) domains. Microinjection of double stranded RNA (dsRNA) corresponding to two disparate portions of the gene resulted in decreased LlIAP mRNA quantities relative to controls. Both nymphs and adult specimens injected with IAP dsRNA exhibited significantly reduced lifespan compared with those injected with non‐insect dsRNA (eGFP). Thus, RNAi‐mediated knockdown of LlIAP expression has been correlated with a lethal phenotype in adults and nymphs.     

Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants

RNA interference (RNAi) using short interfering RNAs (siRNAs) has been widely explored for the suppression of intracellular viral target mRNAs. On the basis of our previous work with stable silencing of Tomato leaf curl virus, in vivo by the antisense replicase gene (AC1) of the virus and characterizing AC4, as a small RNA regulator, besides its role in pathogenicity, we used four different plasmid vector-based siRNA generation strategies to silence viral genes (AC1 and AC4) of tomato leaf curl viruses. The RNAi target sequence were chosen from DNA A of the Tomato leaf curl virus (ToLCV) on the basis of conserved regions in AC1 with an overlapping sequences of the AC4 gene. Different hairpin RNA-mediated strategies like antisense, self-complementary inverted repeats, intron-spliced hairpin RNAs, and small hairpin RNAs were deployed for efficient and predictable resistance to the viruses. Here we present that appropriately designed siRNAs not only prevents RNAi suppression but also help in developing trait-stable transgenics. These strategies imply that ToLCV rep-driven RNAi, targeting AC4 and conserved viral sequences, provides a promising approach to suppress a wide spectrum ToLCV infection in the tomato.     

Down-regulation of IGF-IR using small, interfering, hairpin RNA (siRNA) inhibits growth of human lung cancer cell line A549 in vitro and in nude mice

Type I insulin-like growth factor receptor (IGF-IR), which is frequently overexpressed in a variety of human cancers including lung cancer, mediates cancer cell proliferation and tumor growth. In this study, we used a human U6 promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to silence IGF-IR gene expression in the human lung cancer cell line A549, and then evaluate its effects on apoptosis, apoptosis-related gene expression, and the growth of tumor cells in vitro and in nude mice. IGF-IR expression levels were found to markedly decrease in cells transfected with a plasmid expressing hairpin siRNA for IGF-IR (by more than 78.9%). Down-regulation of IGR-IR concomitantly accompanied reduction of bcl-2 as well as pERK and pAkt levels, activation of caspase-3, apoptosis and growth inhibition of A549 cells in vitro. Direct intratumoral injections of plasmid DNA expressing hpRNA for IGF-IR significantly regressed pre-established tumors in nude mice. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating lung cancer.     

Enzymatic production and expression of shRNAmir30 from cDNAs

RNA interference (RNAi) is an important tool for studying gene function and genetic networks. Double-stranded RNA (dsRNA) triggers RNAi that selectively silences gene expression mainly by degrading target mRNA sequences. Short interfering RNA, short hairpin RNA (shRNA), long dsRNA, and microRNA-based shRNA (shRNAmir) are four different types of dsRNA that have been widely used to silence gene expression in cultured cells, tissues, organs, and organisms. Long dsRNAs are usually 200–500 nucleotides in length and can selectively suppress expression of target genes in Caenorhabditis elegans and Drosophila but not in mammals due to unwanted non-specific knockdown. Thus, multiple attempts have been made to synthesize, express, and deliver short dsRNAs that specifically silence target genes in mammals. We describe a method for constructing an RNAi library by converting cDNAs into shRNAmir30 sequences by sequential treatment with different enzymes and affinity purification of biotin- or digoxygenin-labeled DNA fragments. We also developed a system to generate stable cell lines that uniformly express shRNAmir30s and fluorescence reporters by Cre recombinase-dependent site-specific recombination. Thus, combined with the RNAi library, this system facilitates screening for potent RNAi sequences that strongly suppress expression of target genes.     

基于Semliki森林病毒复制子的新型RNAi质粒载体的构建

目的:以semliki森林病毒复制子为基础,构建一类可迅速高效表达shRNA的新型RNAi载体。方法:以Semliki森林病毒衍生的复制子载体pSFV1为骨架,用CMV IE启动子替换SP6启动子并在3′-UTR下游插入SV40 polyA转录终止子,在原26S亚基因组启动子后插入带有相应改良多克隆位点的shRNA表达元件,同时加入抗新霉素选择复合体,并去掉3′-UTR的重复序列。所获载体用于沉默EGFP基因,通过体外细胞转染、病毒颗粒制备、荧光显微镜观察、RT-PCR分析等初步验证、评估其效果。结果:构建了基于Semliki森林病毒复制子的新型RNAi质粒载体pSFV-RNAi Ready。经体外实验初步证实,该载体直接转染细胞,或与辅助载体共转染,制备成具有感染能力的重组病毒颗粒后使用,均可高水平表达shRNA,沉默目的基因。其中使用病毒颗粒抑抑效率可高达90%以上。结论:该载体的成功构建,可望显著拓宽SFV载体的应用范围,丰富RNAi实施手段,并用于相关科学研究及基因药物技术开发。     

RNAi technology: a new platform for crop pest control

The insect pests are big threat in meeting the food demands for future generation. The present pest control strategies, including the existing transgenic approaches show certain limitations and are not completely successful in limiting the insect pests. However, the sequence-specific gene silencing via RNA interference (RNAi) holds a great promise for effective management of agricultural pests. RNAi is naturally occurring conserved process responsible for gene regulation and defense against pathogens. The efficacy of RNAi varies among different insect orders and also depends upon various factors, including the target gene selection, method of dsRNAs delivery, expression of dsRNAs and presence of off-target effects. RNAi-mediated silencing of different insect genes involved in various physiological processes was found to be detrimental to insects growth, development and survival. In this article, we have reviewed the potential of RNAi-based strategies for effective management of insect pests. We have also discussed the various parameters, which are to be considered for host-induced RNAi-mediated control of insect pests without producing any effect on non-target organisms and environment.     

人骨保护素-热休克蛋白70融合蛋白在昆虫细胞中的表达和活性分析

目的:利用BaculoDirect杆状病毒表达系统融合表达人OPG功能片段p22-194和分枝杆菌HSP70 p111-125基因,并鉴定重组蛋白及其生物学活性。方法:将编码人OPG功能片段和分枝杆菌HSP70功能片段基因克隆至杆状病毒转座载体,将重组转座载体与BaculoDirectTM Linear DNA进行LR重组连接反应,构建出重组杆状病毒DNA,转染Sf9昆虫细胞,获得重组病毒。在Sf9细胞中进行表达,并对表达产物进行SDS-PAGE电泳、Western blotting分析,用Ni柱纯化。采用破骨细胞生成抑制试验和抑炎试验鉴定表达产物的生物学活性。结果:重组病毒在感染昆虫细胞后48h开始出现一相对分子质量为28 kDa大小的特异条带,感染后72～96 h蛋白量达到高峰。破骨细胞生成抑制实验及抑炎试验结果显示,重组蛋白能明显抑制破骨细胞的生长和分化,同时亦具有抑制炎症反应的作用。结论:利用杆状病毒表达系统在昆虫细胞中成功表达OPG-HSP70融合蛋白,该融合蛋白具有抑制破骨细胞生成和抑制炎症反应生物学活性。     

快速直视筛选结缔组织生长因子特异RNA干扰序列

利用一种快速简便经济直接的筛选特异RNA干扰序列的方法,筛选结缔组织生长因子（CTGF）基因特异RNA干扰序列.设计合成分别含EcoRⅠ和HindⅢ酶切位点的引物,以pTM1-CTGF为模板PCR获得CTGF基因,定向克隆入pSOS-HUS质粒得到质粒pSOS-HUS-CTGF. 质粒pSOS-HUS-CTGF经过SfiⅠ酶切后分别与5条CTGF干扰序列和1条随机对照序列克隆获得pSOS-CTGFi1,pSOS-CTGFi2,pSOS-CTGFi3,pSOS-CTGFi4,pSOS-CTGFi5,pSOS-CTGFicontrol.质粒转入HEK293细胞中,于第1、3、5、7 d倒置荧光显微镜照相,IPP图像分析软件测定光强度值.5种CTGF特异RNA干扰载体的相对光强度分别为0.24%、41.47%、2.93%、20.40%和28.85%. 特异性序列site 1和site 3的沉默效果最好. pSOS-HUS系统采用双向启动子,通过绿色荧光蛋白下调水平来衡量干扰序列的沉默效果具有可靠直观重复性好的优点.利用该方法成功筛选了高沉默效果的CTGF特异RNA干扰序列.