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Results for “RNA interference”

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PubMed2005

Silencing the cardiac potassium channel Kv4.3 by RNA interference in a CHO expression system.

RNA interference (RNAi) is a powerful technique for gene silencing, in which the downregulation of mRNA is triggered by short RNAs complementary to a target mRNA sequence, with consequent reduction of the encoded protein. The aim of this study was to test the effects of silencing the expression of the cardiac potassium channel Kv4.3 in a heterologous expression system, in order to investigate the effect of RNAi on channel properties. A Chinese hamster ovary cell line stably expressing Kv4.3 and the accessory beta-subunit KChIP2 was transfected with small-interfering RNAs (siRNAs) targeting Kv4.3. Effects of RNAi were monitored at the mRNA, protein, and functional levels. Real-time PCR and immunofluorescence staining revealed significant reduction of Kv4.3 mRNA and protein expression. These results were confirmed by functional patch-clamp measurements of the transient outward current (I(to)) which was reduced up to 80% by RNAi. We conclude that the use of siRNAs reagents for post-transcriptional gene silencing is a new effective method for the reduction of the expression and function of different ionic channels which may be adapted for studying their role also in native cells.

Animals↗

PubMed2003

Duplexes of 21-nucleotide RNAs mediate RNA interference in differentiated mouse ES cells.

RNA interference (RNAi) is a novel phenomenon that can induce post-transcriptional gene silencing (PTGS) both in animals and plants. RNAi is effective in suppressing specific gene expression in the early mouse embryonic cells and in undifferentiated embryonic stem (ES) cells. In this study, we demonstrate that dsRNA is effective in inducing PTGS in differentiated ES cells: CD34+ embryoid body (EB) cells, as confirmed by western blot and immunocytochemical staining. PU.1 is a key transcription factor in myeloid differentiation. Undifferentiated ES cells do not express PU.1; however it is expressed when ES cells differentiate into EBs. PU.1 could be suppressed by the specific PU.1 dsRNA, but not non-specific Lamin A/C dsRNA, in the CD34+ EB cells when they were induced to myeloid differentiation in the presence of GM-CSF and IL-3. As a consequence, the level of expression of CD115 (M-CSF receptor), one of the downstream genes regulated by PU.1 is decreased in PU.1 dsRNA treated CD34+ EB cells, but not in Lamin A/C dsRNA treated cells. To explore this phenomenon in other myeloid gene, we also found that C/EBPalpha gene could be knocked down by C/EBPalpha dsRNA. Our finding demonstrates that RNAi is effective in inhibiting specific gene expression in differentiated ES cells. RNAi offers a new methodology for study of hematopoietic regulation using ES cell differentiation.

Animals↗

PubMed2004

Use of RNA interference libraries to investigate oncogenic signalling in mammalian cells.

Over the past decade, 'RNA interference' has emerged as a natural mechanism of silencing of gene expression. This ancient cellular antiviral response can be manipulated to provide an effective research tool to knock down the level of expression of selected target genes, providing a very powerful new method for the analysis of cell signalling pathways. Systematic silencing of genes on a genome-wide scale using large rationally designed libraries targeting many thousands of genes provides a novel functional genomics approach to the investigation of many aspects of mammalian cell behaviour, including oncogenic transformation. Here, the different approaches taken to use RNA interference libraries to study the cancer phenotype will be considered, including both selective and high throughput screens and the use of both vector-based and synthetic oligonucleotide-based methods for inducing RNA interference. The advantages and drawbacks of the competing methodologies will be discussed. RNA interference library technology holds great promise for enabling somatic cell genetics in tissue culture systems. Whether it can provide significant new insights into cancer will be its greatest challenge.

Animals↗

PubMed2006

Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus delivery.

RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral strategy. In order to suppress hepatitis B virus (HBV) expression and replication, a retrovirus-based RNAi system was developed, which utilized the U6-RNA polymerase III (Pol III) promoter to drive efficient expression and deliver the HBV-specific short hairpin RNAs (shRNAs) in HepG2.2.15 (2215) cells. In this system, the retrovirus vector with a puromycin selection marker was integrated into the host cell genome and allowed stable expression of shRNAs. In Puro-resistant 2215 cells, the levels of both HBV protein and mRNA were dramatically reduced by over 88% and HBV replication was suppressed. The results demonstrated that retrovirus-based RNAi technology will have foreseeable applications both in experimental biology and molecular medicine.

Gene Expression Regulation, Viral↗

PubMed2005

RNA interference and plant parasitic nematodes.

RNA interference (RNAi) has recently been demonstrated in plant parasitic nematodes. It is a potentially powerful investigative tool for the genome-wide identification of gene function that should help improve our understanding of plant parasitic nematodes. RNAi should help identify gene and, hence, protein targets for nematode control strategies. Prospects for novel resistance depend on the plant generating an effective form of double-stranded RNA in the absence of an endogenous target gene without detriment to itself. These RNA molecules must then become available to the nematode and be capable of ingestion via its feeding tube. If these requirements can be met, crop resistance could be achieved by a plant delivering a dsRNA that targets a nematode gene and induces a lethal or highly damaging RNAi effect on the parasite.

Animals↗

PubMed2005

An antiviral role for the RNA interference machinery in Caenorhabditis elegans.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by exogenous dsRNA. In plants an RNAi-like mechanism defends against viruses, but the hypothesis that animals possess a similar natural antiviral mechanism related to RNAi remains relatively untested. To test whether genes needed for RNAi defend animal cells against virus infection, we infected wild-type and RNAi-defective cells of the nematode C. elegans with vesicular stomatitis virus engineered to encode a GFP fusion protein. We show that upon infection, cells lacking components of the RNAi apparatus produce more GFP and infective particles than wild-type cells. Furthermore, we show that mutant cells with enhanced RNAi produce less GFP. Our observation that multiple genes required for RNAi are also required for resistance to vesicular stomatitis virus suggests that the RNAi machinery functions in resistance to viruses in nature.

Animals↗

PubMed2006

RNA interference and ion channel physiology.

RNA interference (RNAi), through expression of small, double-stranded RNAs or short hairpin RNAs, produces sequence-specific mRNA degradation and decreased gene expression. Since its discovery in 1998 (Fire et al., 1998, Nature 391, 806-811), RNAi has rapidly become one of the most widely used technologies for exploring gene function in eukaryotic cells. Although the topic of RNAi has been the subject of a large number of excellent reviews, the focus of this article is on its application to the study of ion channel physiology in animal cells. In this regard, RNAi has provided definitive identification of ion channel subtypes responsible for both basal and stimulated ion conduction across the plasma membrane of several cell types. The approach has been particularly effective in identifying and establishing the contribution of auxiliary subunits and regulatory proteins to the overall function of ion channel complexes. Moreover, selective knockdown of ion channel expression has been a valuable means of demonstrating roles in the development of specific cell domains and in the normal growth of certain cell types. In this review, a brief description of the general mechanism of RNAi is presented, followed by a discussion of some important considerations for the in vitro application of this technology and in producing transgenic animals as models for human disease. We then describe several examples of where RNAi has been used to investigate the physiological role of ion channels in cells from model organisms (Caenorhabditis elegans and Drosophila melanogaster) and in mammalian cells.

Animals↗

PubMed2006

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference.

BACKGROUND: Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms. RESULTS: We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain. CONCLUSION: A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.

Animals↗

PubMedDate not supplied

Does RNA interference have a future as a treatment for HIV-1 induced disease?

RNA interference has recently emerged as an effective way to block the expression of specific messenger RNAs in eukaryotic cells. Using this approach, it has proven possible to block the replication of HIV-1 in cultured cells using small interfering RNAs targeted to viral sequences or to host messenger RNAs that encode factors critical for virus replication, such as the CCR-5 coreceptor. Unfortunately, the high sequence specificity of RNA interference, combined with the known tendency of HIV-1 to rapidly generate sequence variability, means that HIV-1 variants resistant to individual small interfering RNAs targeted to the viral genome arise rapidly. However, this problem may be circumvented by simultaneously targeting several essential HIV-1 sequences using RNA interference, or by targeting host genes that are essential for virus replication. Thus, RNA interference-based approaches have the potential to prove useful as novel treatments for HIV-1 induced disease, although the problem of how to efficiently deliver small interfering RNA expression vectors, or the small interfering RNAs themselves, to cells susceptible to HIV-1 infection in vivo, remains to be resolved.

HIV Infections↗

PubMed2003

[RNA interference in three ES cell lines from different mouse strains].

RNA interference phenomenon in three different murine ES cell lines (MESPU13, B3, and R1) is reported. A vector(pdsGFP) was used that transcribed hairpin double-stranded RNA of GFP gene to transfect ES cells by using lipofectin. The transient transcription of dsRNA induced RNAi (RNA interference) in the ES cells. That is, the double-stranded RNA of GFP gene potently turned down the expression of the GFP gene. On the hand, the linearized plasmid pdsGFP-puro was electroporated into MESPU13 ES cells, and the expression level of GFP after puromycin screening was turned down obviously in about 30% ES cell clones; and in a few clones, the expression level of GFP was not observed under the fluorescence microscope and GFP mRNA was not detectable by RT-PCR. Further more, another vector (pdsOCT4) was constructed that transcribed double-stranded RNA of OCT-4 gene which is specifically expressed in ES cells. ES cell clones that stably integrated the vector were screened after the electrotransfection of the cells with the above construct. 51 random-selected clones were amplified and 48 of them were checked by semi-quantitative RT-PCR. In 11 of them the mRNA of OCT-4 was undetectable by RT-PCR. This means that RNAi can be used to study mammal and human gene's function in ES cell lines from different strain mice.

Animals↗

PubMed2004

RNA interference--small RNAs effectively fight viral hepatitis.

RNA interference (RNAi) is the process of sequence-specific gene silencing, initiated by double-stranded RNA that is homologous in sequence to the target gene. This unique phenomenon has been extensively investigated during the last few years not only in the context of its mechanism and its possible role in the regulation of gene expression and cell function, but also as a potential powerful tool for gene therapy. Targeting essential viral genes or oncogenic alleles are only some of the possible applications of RNAi in the field of gene-directed therapy. This review covers the potential use of RNAi against hepatitis B and hepatitis C viruses, the main pathogens causing chronic liver disease. The major milestones along the discovery of RNAi will also be covered.

Animals↗

PubMed2006

Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.

Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem-loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

Animals↗

PubMed2006

Is RNA interference involved in intrinsic antiviral immunity in mammals?

RNA interference constitutes a key component of the innate immune response to viral infection in both plants and invertebrate animals and has been postulated to have a similar protective function in mammals. This perspective reviews the available data addressing whether RNA interference forms part of the mammalian innate immune response and concludes that the popular hypothesis in favor of that possibility remains far from proven and may not be valid.

Animals↗

PubMed2003

RNA interference: silencing in the cytoplasm and nucleus.

Although the discovery that double-stranded RNA is able to silence gene expression was only made five years ago, methods for experimentally silencing genes have already been extended into a broad diversity of organisms, including human cells. RNA interference has also been discovered to function in physiological gene silencing. RNA interference works by causing degradation of targeted mRNAs in the cytoplasm. However, recent results suggest that RNA interference may also silence gene activity in the nucleus by remodeling chromatin and repressing the transcription of targeted genes.

Animals↗

PubMed2004

RNA interference (RNAi) with RNase III-prepared siRNAs.

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have varying silencing capacities, several different siRNAS typically must be screened to obtain a region that will effectively silence the gene of interest. However, RNA interference with synthetic siRNA is inefficient and cost-intensive, especially for large, functional genomic studies. Here, we describe the use of E. coli endoribonuclease III to cleave double-stranded RNA (dsRNA) into esiRNA (endoribonuclease-prepared siRNA) that can target multiple sites within an mRNA. EsiRNA mediates effective RNA interference with no apparent nonspecific effects in cultured mammalian cells. Since the whole gene can be used at once, screening for an active siRNA for an individual gene is eliminated. Because of its simplicity and potency, this approach is useful for large-scale analysis of mammalian gene function.

Base Sequence↗

PubMed2005

The future of HIV infection: gene therapy and RNA interference.

The description of the mechanism of RNA interference (RNAi) has generated enormous interest in the biomedical field. A previously unrecognized pathway in which small interfering, 21 to 23 mer, double-stranded RNA (siRNA) mediates sequence-specific degradation of mRNA is becoming one the most useful techniques in cell biology and genetics research. Based on the potency, specificity and physiology of RNAi to silence gene expression, much is expected from its use as a therapeutic tool. The first evidence of RNAi as a suppressor of HIV replication has already been reported, thus providing a new impetus to the development of molecular or gene therapy approaches to HIV infection.

Adult↗

PubMedDate not supplied

[RNA interference: biology and perspectives of application in biomedicine and biotechnology].

RNA interference (RNAi) is among the most particular mechanisms of gene expression regulation. Besides, small interfering RNAs are significant players in cell defence either from viral infection or retrotransposons. Medical utilization of RNAi gives a handful of ways to cure viral and oncological illnesses. RNA interference, also, represents a useful tool for research, because it allows quick production of monogene functional knockouts. In this review we describe the most recent conceptions about RNAi mechanisms and actual approaches for it's usage.

Animals↗

PubMed2006

RNA interference inhibits hepatitis B virus gene expression and replication in HepG2-N10 cells.

OBJECTIVE: RNA interference (RNAi) refers to the phenomenon of sequence-specific degradation of homologous mRNA induced by double-stranded RNA. It has been successfully utilized to down-regulate endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, the effect of vector-based small interfering RNA (siRNA) promoted by pSilencer2.0-U6 inhibit hepatitis B virus (HBV) replication in cell culture was evaluated. METHODS: Three fragments of short nucleic acids, respectively, targeting on S, X and C region of HBV genome were inserted into pSilencer vectors after they were annealed with their partly antisense strands. The recombination plasmids were pS, pX and pC. These expression plasmids were transfected into HepG2-N10 cells, a cell line which stably expresses hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg) and adw2 subtype Dane particles. The effect of RNAi was evaluated from the changes of DNA, RNA and protein levels. Viral antigens were measured by ELISA. Viral mRNA was analyzed by RT-PCR. The covalent closed circular DNA and genome DNA of HBV secreted into the culture media were measured by quantitative real-time PCR. Analysis of variance was performed for the results. RESULTS: Vector-based RNA interference could potently reduce HBsAg (pS vs pN: 47%, pX vs pN: 30%, and pC vs pN: 25%, P < 0.001) and HBeAg (pX vs pN: 57% and pC vs pN: 66%, P < 0.001) expression in cell culture. Furthermore, RT-PCR analysis showed that viral mRNAs were effectively degraded, thus eliminating the messengers for protein expression as well as templates for reverse transcription (pS and pC vs pN, P < 0.001; pX vs pN, P = 0.003). Quantitative real-time PCR analysis of HBV DNA revealed that vector-based RNA interference can inhibit HBV replication efficiently (pS, pX and pC vs pN, P < 0.001). CONCLUSIONS: Our results indicate that RNAi can inhibit HBV gene expression and replication, and it might have the potential to revolutionize the treatment of HBV.

Cell Line↗