Specific gene silencing using small interfering RNAs in fish embryos

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.     

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.     

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.     

c-fos antisense RNA blocks expression of c-fos gene in F9 embryonal carcinoma cells

To study the function of proto-oncogene c-fos, we prepared an antisense plasmid that expresses in mammalian cells c-fos antisense RNA which is complementary to the endogenous c-fos mRNA. Upon transfection into undifferentiated F9 EC cells, the antisense plasmid directed constitutive expression of a large amount of c-fos antisense RNA. These cells were very low in the basal level of c-fos message and were unable to induce c-fos message when stimulated with interferon or phorbol ester. The failure to induce c-fos message led to the blockade of c-fos protein expression in these cells. Thus, these cells represented a c-fos defective phenotype. The blockade of c-fos gene expression seen in antisense-cells could be caused by rapid degradation of the c-fos message, since c-fos mRNA expression was rescued in these cells when treated with protein synthesis inhibitor, cycloheximide. We found that expression of c-myc gene was down-regulated in c-fos antisense-cells: Although control undifferentiated F9 cells constitutively expressed a high level of c-myc message, the antisense cells had a much lower amount of c-myc mRNA. Since p53 and heat shock gene 70 were expressed at comparable levels in control and antisense cells, c-myc gene expression appears to be regulated by c-fos gene in F9 EC cells. Lastly, these antisense cells grew as rapidly as control F9 cells and underwent differentiation after retinoic acid treatment, indicating that c-fos expression is not a prerequisite for differentiation of F9 cells.     

RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.     

Potato virus Y mRNA expression knockdown mediated by siRNAs in cultured mammalian cell line

RNA interference (RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes. The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion. In our study, a 480bp fragment of the capsid protein gene of potato virus Y (CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro, as the CP gene interferes with viral uncoating, translation and replication. A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells. CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs. Six biological replicates were performed in this study. In our findings, one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%–90%.     

A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells

We describe a rapid cell-based genetic screen using fission yeast for identifying efficient gene suppression constructs (GSCs) from large libraries (10⁵) for any target sequence for use in human cells. In this system, target sequences are fused to the 5′ end of the lacZ reporter gene and expressed in yeast. Random fragment expression libraries derived from the target sequence are screened in the fusion gene-expressing strain using the lacZ gene-encoded colony color phenotype. We demonstrate the utility of this screening assay by identifying a range of different GSCs for the fission yeast ura4 gene and human c-myc and Chk1 sequences, including rare efficient suppressors. GSCs specific for c-myc were shown to regulate expression of both a c-myc–lacZ fusion gene and the endogenous c-myc gene in human cells.     

RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays

RNA interference (RNAi) is a process in which double-strand RNA (dsRNA) directs the specific degradation of a corresponding target mRNA. The mediators of this process are small dsRNAs, of ~21 bp in length, called small interfering RNAs (siRNAs). siRNAs, which can be prepared in vitro in a number of ways and then transfected into cells, can direct the degradation of corresponding mRNAs inside these cells. Hence, siRNAs represent a powerful tool for studying gene functions, as well as having the potential of being highly specific pharmaceutical agents. Some limitations in using this technology exist because the preparation of siRNA in vitro and screening for siRNAs efficient in RNAi can be expensive and time-consuming processes. Here, we demonstrate that custom oligonucleotide arrays can be efficiently used for the preparation of defined mixtures of siRNAs for the silencing of exogenous and endogenous genes. The method is fast, inexpensive, does not require siRNA optimization and has a number of advantages over methods utilizing enzymatic preparation of siRNAs by digestion of longer dsRNAs, as well as methods based on chemical synthesis of individual siRNAs or their DNA templates.     

A Simplified and Versatile System for the Simultaneous Expression of Multiple siRNAs in Mammalian Cells Using Gibson DNA Assembly

RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.     

Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system

Recently, small interfering RNAs (siRNAs) have become a powerful and widely used tool for the analysis of gene function in mammalian cells. Here we report that the microinjection of an siRNA expression vector into the nucleus is an efficient and powerful method of specific gene silencing in pre-implantation mouse embryos. We used this method to examine the expression of two genes EGFP and Oct4. Vectors encoding siRNAs targeted against EGFP or Oct4 were injected into the pronucleus or nucleus of zygotes, which were then cultured until the blastocyst stage. When the effects of RNAi were examined in blastocyst stage eggs, there was robust inhibition of the gene product in a concentration-dependent manner at both the mRNA and the protein level. The expression of other endogenous genes was not affected, showing the specificity of the vector-mediated RNAi. In addition, this method was effective for inhibiting maternally expressed mRNA. To demonstrate that RNAi of Oct4 induced a similar phenotype to that of Oct4-null embryos, the blastocysts were further cultured in ES medium. After the fourth day of culture, the embryos either had outgrown only a layer of trophoblast cells or showed developmental arrest at the blastocyst stage (>90%). Moreover, concomitant with Oct4 suppression at the blastocyst stage, we observed inhibition of Fgf4, a gene that is known to be induced downstream of Oct4 expression. Taken together, these results demonstrate that the use of siRNA expression vector is a powerful way to achieve gene silencing in the pre-implantation stage embryo.     

Subtype- and species-specific knockdown of PKC using short interfering RNA

RNA interference (RNAi), the targeted mRNA degradation induced by double-stranded RNA (dsRNA), is a powerful tool for analyzing gene function in many organisms. Recently, it has been shown that RNAi is also applicable to cultured mammalian cells by using short interfering RNA (siRNA) [Nature 411 (2001) 494]. To examine whether this siRNA method is useful for analyzing the subtype-specific functions of protein kinase C (PKC), we first prepared siRNAs which target human alphaPKC and human deltaPKC and applied them into mammalian cells to suppress the expression of endogenous alphaPKC and deltaPKC, respectively. Each siRNA for alpha or deltaPKC specifically suppressed the endogenous expression of corresponding PKC subtype in human-derived cell lines such as HEK-293 and HeLa cells, but not in cells derived from rat species. The suppression level of deltaPKC reached maximum 48-72h after the transfection of siRNA. In addition, the siRNA targeting rat deltaPKC suppressed endogenous and exogenous rat deltaPKCs but not human deltaPKC, suggesting that siRNAs targeting PKCs effectively knocked down endogenous/exogenous PKCs in mammalian cells, in subtype- and species-specific manner. Furthermore, we also developed the method to discriminate the siRNA-transfected cells using the antibody recognizing thymine dimer. Our present results strongly suggest that siRNA method enable us to examine the subtype-specific function of PKC, not only by knockdown of the endogenous target PKC subtype, but also by subsequent compensation with the exogenous corresponding wild/mutant PKC derived from other species.     

Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA

RNA interference (RNAi) is a RNA-mediated sequence-specific gene silencing mechanism. Recently, this mechanism has been used to down-regulate protein expression in mammalian cells by applying synthetic- or vector-generated small interfering RNAs (siRNAs). However, for the evaluation of this new knockdown technology, it is crucial to demonstrate biological consequences beyond protein level reduction. Here, we demonstrate that this new siRNA-based technology is suitable to analyse protein functions using the phosphatidylinositol (PI) 3-kinase signal transduction pathway as a model system. We demonstrate stable and transient siRNA-mediated knockdown of one of the PI 3-kinase catalytic subunits, p110β, which leads to inhibition of invasive cell growth in vitro as well as in a tumour model system. Importantly, this result is consistent with loss-of-function phenotypes induced by conventional RNase H-dependent antisense molecules or treatment with the PI 3-kinase inhibitor LY294002. RNAi knockdown of the downstream kinases Akt1 and Akt2 does not reduce cell growth on extracellular matrix. Our data show that synthetic siRNAs, as well as vector-based expression of siRNAs, are a powerful new tool to interfere with signal transduction processes for the elucidation of gene function in mammalian cells.