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.     

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 interfering approach for clarifying the PPARgamma pathway using lentiviral vector expressing short hairpin RNA

Peroxisome proliferator-activated receptor γ (PPARγ) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARγ also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARγ expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARγ-knockdown method will serve to clarify the PPARγ pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.     

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific fashion.Small interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells.Here,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant tissue.Our results indicate that transiently expressed shRNA efficientlyinterfered with TMV infection.The interference observed is sequence-specific,and time-and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated tobamovirus.In order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation.Ourresults support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.     

A technique to enzymatically construct libraries which express short hairpin RNA of arbitrary stem length

Short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) usually used for RNA interference (RNAi) are double-stranded RNAs (dsRNAs) of 21 base pairs. However, siRNAs and shRNAs of longer stem length have been reported to show more potent gene silencing. Here, we report a new technique to enzymatically construct shRNA libraries containing clones from firefly luciferase cDNA and Jurkat cDNA. The technique allowed the efficacious generation of shRNAs of arbitrary stem length as desired, providing the clones which potently silenced the specified gene expression and presenting a high efficiency rate of gene silencing. Our results indicate that the technique permits the rapid, efficient, and low-cost preparation of genomewide shRNA expression libraries not only for humans and mice but also for sorts of biological species and that the relevant libraries are applicable for the search of genes related to phenotype changes and of new targets for drug discovery.     

转病毒来源发夹RNA小麦表现对大麦黄矮病毒的抗性

将大麦黄矮病毒GPV株系的复制酶基因片段和CP基因片段构建成可在植物细胞内表达含有双链复制酶RNA(茎)和反义CP RNA(环)的复合发夹RNA结构, 希望能够诱发植物体针对病毒的RNA干扰作用, 从而达到抗病毒目的。利用基因枪法将该结构导入小麦幼胚愈伤组织细胞后, 通过在幼苗再生阶段进行以叶片为模板的快速PCR来加速阳性植株的筛选过程, 最终共获得基因组整合有外源基因的小麦再生植株21株。对再生植株接种不同剂量的病毒, 其中9株对BYDV-GPV有低度抗性, 表现在低接毒量时无症状, 接毒量提高时发病且严重; 6株具中度抗性, 表现在低接毒量时无症状, 接毒量提高时局部有不严重症状; 6株具高度抗性, 两种情况下均无症状。抗性实验结果表明, hpRNA介导对BYDV的抗性可能受到BYDV含量的影响, 具有剂量效应的特点。     

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.     

COS-7细胞中小发卡RNA介导的RNA干涉

利用U6启动子转录小发卡RNA介导的RNA干涉是最近发展起来的在哺乳动物细胞中特异性抑制指定基因表达的新技术,已有实验证明它在小鼠畸胎瘤P19等细胞系中具有强烈的抑制基因表达的作用。本文对COS－7细胞系中U6启动子转录GFP基因的小发卡RNA介导的RNA干涉现象进行了研究,结果表明：U6启动子转录的小发卡RNA具有RNA干涉作用,即可以在COS－7细胞中特异性地抑制含有对应序列的基因GFP的表达,这一结果为今后在COS－7细胞系中利用RNA干涉技术研究目的基因的功能奠定了基础。     

Optimization of a genome-wide disordered lentivector-based short hairpin RNA library

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15–20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro ^R, copGFP, or H-2K ^k as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.     

各种RNA干扰库的构建及比较

不同种类的RNA干扰库已经被用于基因功能的研究之中,根据其构建方式和分子形式的不同,可将RNA干扰库分为4种类型,即化学合成的小干扰RNA（siRNA）库、化学合成的小发夹RNA（shRNA）表达库、酶切法构建的siRNA库和酶切法构建的shRNA表达库。简要介绍上述RNA干扰库的构建方法及其特点和局限。     

RNA-binding proteins in RNA interference

Short RNAs (21–27 nt) silence genes that contain homologous nucleotide sequences; this is known as RNA silencing. This review considers the generation of short RNAs from their precursors: double-stranded RNAs, capable of inducing RNA interference, and hairpin RNAs, whose processing yields microRNAs, as well as the properties of RNA-binding domains that were initially identified in proteins operating in RNA interference. The interactions between these domains and known RNA-binding modules within proteins involved in RNA interference and microRNA generation are described.     

shRNA Transcribed by RNA Pol II Promoter Induce RNA Interference in Mammalian Cell

RNA interference is a powerful tool for gene functional analysis in mammals. Permanent gene suppression can be achieved by siRNAs as stem-loop precursors transcribed from RNA Pol III promoter such as H1 and U6 based on vector. This approach, however, has a major limitation: inhibition can not be controlled in a time or tissue specific manner because the RNA Pol III promoter is not time or tissue specific. To overcome these limitations, we designed a strategy that allows synthesis of small hairpin RNAs in a GFP-fused form mediated by RNA Pol II promoter CMV to efficiently and specifically knock down expression of both exogenous and endogenous genes in mammalian cells. As assayed by both fluorescence observing and quantitative RT-PCR, the protein and mRNA products of exogenous gene RFP were efficiently and specifically inhibited; quantitative RT-PCR and western blotting results respectively demonstrated that endogenous lamin B2 mRNA and protein was suppressed without global down-regulation of protein synthesis. Furthermore, GFP-fused shRNA efficacy for RNAi is dependent on target position based on this vector system. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in mammalian system. Jing Yuan, Xiaobo Wang and Ning Li - These authors contributed equally to this work.     

Applications of RNA interference high-throughput screening technology in cancer biology and virology

RNA interference (RNAi) is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi high-throughput screening (HTS) has opened an exciting frontier to systematically study gene function in mammalian cells. This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight. Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.     

Phenotypic inheritance induced by hairpin RNA in Drosophila

Phenotypic inheritance induced by RNA has been documented in mouse and Caenorhabditis elegans. Here we report a similar inheritance in Drosophila. Mutant phenotypes of eye defects and antenna duplication generated from the crossing of one RNA interference （RNAi） transgenic line harboring one hairpin RNA transgene with a GAL4 driver line were inherited independently of the GAL4 driver. Hairpin RNA injection experiments demonstrated that the hairpin RNA could induce heritable mutant-like phenotypes on the eye and antenna. The penetrance of mutant phenotypes was reduced when the mutants were crossed to agol and piwi mutants. Our data suggest that hairpin RNA can induce phenotypic inheritance in Drosophila.