Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression

RNA interference (RNAi) mediated by DNA-based expression of short hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research.     

survivin启动子驱动的shRNA在宫颈癌SiHa细胞中的特异性表达

研究survivin启动子驱动的小发夹RNA(shRNA)在宫颈癌SiHa细胞中的特异性表达.PCR扩增survivin启动子,构建以survivin启动子驱动增强绿色荧光蛋白(EGFP)表达的真核表达载体pEGFP-C1/Surv,并检测survivin启动子的活性;构建针对EGFP的shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6,并检测shRNA在U6启动子驱动下的干扰效果;用有活性的survivin启动子取代pGenesil-1-EGFP-shRNA/U6载体中的U6启动子,从而获得新的shRNA真核表达载体pGenesil-1-EGFP-shRNA/Surv;将pGenesil-1-EGFP-shRNA/Surv转染到宫颈癌SiHa细胞及正常人脐静脉内皮HuVEC细胞,观察EGFP在两种细胞中的表达变化.结果表明,成功扩增survivin启动子并验证该启动子具有活性;成功构建EGFP shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6,并验证shRNA在U6启动子驱动下具有较好的干扰效果;成功构建pGenesil-1-EGFP-shRNA/Surv载体,分别转染SiHa及HuVEC细胞,在SiHa 细胞中观察到较少的绿色荧光蛋白表达,而在HuVEC细胞中观察到较多的绿色荧光蛋白表达.survivin启动子能驱动shRNA在宫颈癌SiHa细胞中特异性表达,而在正常HuVEC细胞中不表达.     

Cloning and functional analysis of sheep U6 promoters

Gene silencing mediated by small interfering RNA has become a powerful biological tool for the regulation of gene expression. In order to develop an effective short hairpin RNA (shRNA) expression vector, specifically for use in sheep species, we have identified two sheep U6 promoters based on the highly conserved polymerase III promoter elements. Promoter activity was measured by U6 promoter-driven shRNA to suppress enhanced green fluorescent protein (EGFP) expression. The knock down assay demonstrated that the two sheep U6 promoters and mouse U6 promoter induced a similar level of EGFP knockdown. These results suggest that the two sheep U6 promoters could efficiently drive shRNA expression for gene silencing and may have applications in RNAi-based sheep research.     

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.     

Altering Cellular Signaling Pathways Enhance Gene Silencing Activity of shRNA,shRNA.Ribozyme,and shRNA.Antisense in Neuroblastoma Cells

1. RNA interference (RNAi) is a multicomponent machinery that operates in a sequence-specific manner to repress the expression of genes in most eukaryotic cells.2. Here we wanted to investigate in a murine neuroblastoma cell line (NBP2) (a) if replacement of the loop of the short hairpin RNA (shRNA) with a hammerhead ribozyme (shRNA.RZ) or an antisense oligonucleotide (shRNA.AS) would affect the efficacy of gene suppression, and (b) if activation or inhibition of signaling pathways would enhance the efficacy of shRNA, shRNA.RZ, and shRNA.AS complex in gene silencing.3. We used U6-driven expression of these shRNAs to target either a short-lived green fluorescent protein (d2EGFP) or an endogenous cyclophilin A (CyP-A) gene in a d2EGFP expressing NBP2 cell line (NBP2-PN25).4. Activation of the cAMP signaling pathway or inhibition of phosphatidylinositol 3-kinase (PI3K) enhanced the efficacy of shRNA and shRNA.RZ complex in reducing the expression of d2EGFP shRNA.RZ complex was as efficacious as shRNA in reducing the expression of d2EGFP and CyP-A shRNA.AS complex showed a slightly lower efficacy than shRNA alone in decreasing d2EGFP expression. In contrast, the U6-driven hammerhead ribozyme targeted to d2EGFP showed no gene silencing activity.5. This report describes novel strategies of modifying shRNA and altering signaling pathways to affect siRNA-mediated gene silencing in a neuronal cell line.     

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 .     

Characterisation and comparison of the chicken H1 RNA polymerase III promoter for short hairpin RNA expression

The U6 and 7SK RNA polymerase III promoters are widely used in RNAi research for the expression of shRNAs. However, with their increasing use in vitro and in vivo, issues associated with cytotoxicity have become apparent with their use. Therefore, alternative promoters such as the weaker H1 promoter are becoming a popular choice. With interest in the chicken as a model organism, we aimed to identify and characterise the chicken H1 promoter for the expression of shRNAs for the purpose of RNAi. The chicken H1 promoter was isolated and sequence analysis identified conserved RNA polymerase III promoter elements. A shRNA expression cassette containing the chicken H1 promoter and shRNA targeting enhanced green fluorescent protein (EGFP) was developed. An RNAse protection assay confirmed activity of the promoter determined by the detection of expressed shRNAs. Comparison of the H1 promoter to the chicken RNA polymerase III 7SK and U6 promoters demonstrated that expressed shRNAs from the H1 promoter induced gene specific silencing, albeit to lower levels in comparison to both 7SK and U6 promoters. Here we have identified a new tool for RNAi research with specific applications to the chicken. The availability of a RNA polymerase III promoter that drives shRNA expression to reduced levels will greatly benefit in ovo/in vivo applications where there are concerns of cytotoxicity resulting from overexpression of an shRNA.     

利用人U6 snRNA启动子构建RNA干扰质粒载体

RNA干扰技术已经成为基因功能研究等领域的有力工具,构建带有筛选标记的siRNA载体可以在细胞中持续抑制靶基因的表达.为了利用RNAi技术开展生物学研究,在克隆载体pUC19的基础上改造构建了人类细胞小干扰RNA（small interference RNA,siRNA）表达质粒pUC19NU.该质粒具有新霉素抗性标记和真核细胞复制起点,利用连入的人U6 snRNA启动子起始siRNA的转录.以EGFP 和p53为靶基因的干扰实验证明,所构建的siRNA表达质粒可以显著抑制细胞外源性增强绿色荧光蛋白（enhanced green fluorescent protein,EGFP）及细胞内源性p53蛋白的表达,而且抑制效果具有特异性.     

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.     

RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter

Small interfering RNAs (siRNAs) represent RNA duplexes of 21 nucleotides in length that inhibit gene expression. We have used the human gene-external 7S K RNA promoter for synthesis of short hairpin RNAs (shRNAs) which efficiently target human lamin mRNA via RNA interference (RNAi). Here we demonstrate that orientation of the target sequence within the shRNA construct is important for interference. Furthermore, effective interference also depends on the length and/or structure of the shRNA. Evidence is presented that the human 7S K promoter is more active in vivo than other gene-external promoters, such as the human U6 small nuclear RNA (snRNA) gene promoter.     

Schistosoma mansoni U6 gene promoter-driven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules

RNA interference (RNAi) mediated by short hairpin-RNA (shRNA) expressing plasmids can induce specific and long-term knockdown of specific mRNAs in eukaryotic cells. To develop a vector-based RNAi model for Schistosoma mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni. A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 h following transduction with plasmid pXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In a similar fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared with controls. The findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes. Given that the U6 gene promoter drove expression of shRNA from an episome, the findings also indicate the potential of this putative RNA polymerase III dependent promoter as a component regulatory element in vector-based RNAi for functional genomics of schistosomes.     

RNA干扰技术抑制内源性绿色荧光蛋白的表达

目的建立稳定表达绿色荧光蛋白(GFP)的细胞株;构建短发夹RNA(shRNA)表达质粒并观察其对内源性GFP的抑制作用。方法转染pEGFP-N1至HepG2细胞,利用G418筛选获得稳定表达GFP的细胞株(HepG2.GFP);设计合成针对GFP基因的siRNA对应的DNA片段,插入转录载体pTZU6 1,构建shRNA表达载体pSHGFP,转染HepG2.GFP,荧光显微镜观察细胞荧光强度,以western blot检测GFP蛋白水平,以RT-PCR检测mRNA水平。结果利用PCR方法从HepG2.GFP细胞基因组DNA中检测到GFP基因;pSHGFP能够显著抑制该细胞中GFP的表达。结论GFP基因成功整合至HepG2细胞基因组中,pSHGFP能够显著抑制内源性GFP的表达,该系统能够用于RNA干扰机制等研究中。