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At least 667 records · Page 37Linked to original sources
PubMed2006

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype.

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Base Sequence↗
PubMed2003

Gene silencing in chick embryos with a vector-based small interfering RNA system.

In this paper, the use of vector-based RNA interference (RNAi) to specifically interfere with gene expression in chick embryos is reported. In ovo electroporation was carried out to transfer a small interfering RNA (siRNA) expression vector into chick embryos. En2 was chosen for the target gene because the family gene, En1, is expressed in a similar pattern. Four sets of 19-mer sequences were designed with the En2 open reading frame region connected to a sequence of short hairpin RNA (shRNA), which exerts siRNA effects after being transcribed, and inserted into pSilencer U6-1.0 vector. En2 and En1 expression were suppressed by the siRNA whose sequence completely matched En2 and En1. Suppression occurred when the siRNA sequence differed by up to two nucleotides from the target sequence. The sequence that differed by four nucleotides from the target gene did not show siRNA effects. One set that completely matched the En2 target did not show siRNA effects, which may be due to location of the siRNA in the target gene. Thus, multiple sets of shRNA must be prepared if we are to consider. This system will greatly contribute to the analysis of function of genes of interest, because the target gene can be silenced in a locally and temporally desired manner.

Animals↗
PubMed2006

Multiple shRNAs expressed by an inducible pol II promoter can knock down the expression of multiple target genes.

RNA interference (RNAi) has been increasingly used for reverse genetics. Both pol III and pol II promoters have been used to synthesize short hairpin RNA (shRNA) for knockdown of gene expression in mammalian cells and animals. Compared with gene knockout approaches, RNAi has the advantage of being simple, quick, and low cost. Here we describe a method that enhances this advantage where knockdown of expression of multiple genes in the same cells is required. A tetracycline-regulated pol II promoter construct allows the expression of up to three shRNA genes that have been cloned into introns of a transcript bearing green fluorescent protein (GFP) coding sequences. This method may be used to establish stable knockdown cell lines and may also prove useful for investigating gene-gene interactions in transgenic animals.

Animals↗
PubMed2000

RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.

Receptor protein tyrosine phosphatases (RPTPs) are important for growth-cone migration [1-5], but their specific roles have yet to be defined. Previously, we showed that the growth cones of the Comb cell, an embryonic cell in the leech, express high levels of an RPTP called HmLAR2 [6,7]. Here, we report the use of RNA interference (RNAi) to block expression of HmLAR2 in individual Comb cells in the developing embryo. HmLAR2 mRNA levels were reduced in the soma, processes and growth cones of Comb cells injected with double-stranded RNA (dsRNA) for HmLAR2, but no decrease was detected when control dsRNAs were injected. Consistent with this observation, the level of phosphotyrosine increased significantly in the growth cones of Comb cells injected with HmLAR2 dsRNA. Within 24 hours, the growth cones of treated cells showed a distinct collapsed phenotype, with sharp reductions in lamellipodial surface area and in numbers of filopodia. These experiments indicate a key role for LAR-like RPTPs in maintaining the integrity of the growth cone.

Animals↗
PubMed2005

Analysis of T-cell development by using short interfering RNA to knock down protein expression.

We have applied RNA interference (RNAi) technology to the analysis of genes involved in T-cell development, combining a reaggregate fetal thymic organ culture (rFTOC) system with retroviral delivery of short interfering RNA (siRNA) hairpins. The process involves the isolation of murine fetal liver or fetal thymocytes, infection with retroviral particles carrying the construct of interest, followed by reaggregation of the transduced precursors with fetal thymic stroma into lobes. Subsequently, individual lobes are harvested and analyzed for development at various time points. These reaggregate cultures recapitulate most features of T-cell development in vivo, including pre-TCR selection and expansion, positive selection of CD4 and CD8 T cells, and negative selection. In our hands, the combination of retroviral delivery of RNAi and rFTOCs is a quick alternative to conventional knockouts for the analysis of gene function during T-cell development. This chapter describes the methods we have developed to knock down gene expression in T-cell precursors, using retroviral delivery of siRNA hairpins.

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↗
PubMed2006

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing.

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

Animals↗
PubMedDate not supplied

RNA interference--significance and applications.

RNA interference (RNAi) is a post-transcriptional, highly conserved process in eukaryotes that leads to specific gene silencing through degradation of the target mRNA. This mechanism is mediated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The dsRNA is processed into small interfering RNA (siRNA) by an enzyme called Dicer, and the siRNAs are then incorporated into a multi-component RNA-induced silencing complex, which finds and cleaves the target mRNA. In plants and worms, amplification of the silencing signal and cell-to-cell RNAi spreading is observed. The proposed biological roles of RNAi include resistance to viruses, transposons (mainly in plants), and the silencing and regulation of gene expression, particularly during development. In developmental gene control, specific small RNAs (micro RNA and small temporal RNA) are involved, which are processed in the same way as dsRNAs but act at the level of translation. RNAi technology has become a powerful tool in functional genomic analyses and may prove to be a useful method to develop highly specific gene-silencing therapeutics against viral infections and cancer in the future.

Animals↗
PubMed2006

Inhibition of CD147 expression reduces tumor cell invasion in human prostate cancer cell line via RNA interference.

CD147, also named extracelluar matrix metalloproteinase inducer (EMMPRIN), has been proved to be involved in the invasion and metastasis processes of tumor cells in many types of cancers. To determine the role of CD147 in the invasiveness properties of prostate cancer, we successfully downregulated CD147 by RNA interference (RNAi) technology, in PC-3 cell line at high level of CD147 expression. PC-3 cells were transfected with a pSilencer 4.1-CMV neo Vector coding for an RNA composed of two identical 19-nucleotide sequence motifs in an inverted orientation, separated by a 9-bp spacer to form a hairpin dsRNA capable of mediating target CD147 inhibition. Gelatin zymography was employed to determine the effect on reducing secretions of MMP-2 and MMP-9 of the transfected cells. Matrigel invasion assay was performed to evaluate the invasion ability of PC-3 cells in vitro. Our results showed that CD147 expression was significantly inhibited by small interfering RNAs (siRNA) transfectants in PC-3 cells at mRNA and protein levels, which resulted in dramatic reduction of invasion ability in tumor cells. Moreover, downregulation of CD147 resulted in reducing secretions of MMP-2, MMP-9. Taken together, CD147 downregulation by RNAi technology decreases the invasive capability of prostate cancer cells, demonstrating that stable expression of siRNA CD147 could potentially be an experimental approach for prostate cancer gene therapy.

Antigens, CD↗
PubMed2004

Dynamics of gene silencing by RNA interference.

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.

Animals↗
PubMed2006

Telomerase inhibition by an siRNA directed against hTERT leads to telomere attrition in HT29 cells.

Human telomerase is a ribonucleoprotein complex composed of at least the reverse catalytic transcriptase hTERT and RNA component hTR. The enzyme stabilizes telomere length and thereby contributes to unlimited cell proliferation, i.e. immortality. Reactivation of telomerase activity during carcinogenesis is a common hallmark in most human tumor types. Consequently, telomerase is an attractive molecular target toward which to direct cancer therapeutic agents. RNA interference (RNAi) has been shown to be an effective method for inhibiting the expression of a given gene in human cells by targeting with short duplex RNA (short-interfering RNA or siRNA). Thus, we evaluated the ability of siRNAs to inhibit telomerase activity in the HT29 immortal human colorectal adenocarcinoma cell line as a model for colorectal carcinogenesis. By transient expression of a specific siRNA directed against hTERT, we reduced telomerase activity in the transfected cells. Moreover, telomere lengths were reduced in cells stably expressing this particular RNA sequence, cloned as an shRNA into an eukaryotic expression vector. The cell clone that displayed a telomerase-negative phenotype showed dramatically reduced telomere lengths and stopped proliferation. We observed that the vector was integrated into the cell genome and, despite telomere shortening, cells retained their MSI phenotype. We conclude that we have developed a potent telomerase inhibitor leading to cell death.

Adenocarcinoma↗
PubMed2005

In vitro and in vivo suppression of GJB2 expression by RNA interference.

Mutations in GJB2 (gap junction protein, beta-2) are the major cause of autosomal recessive non-syndromic hearing loss. A few allele variants of this gene also cause autosomal dominant non-syndromic hearing loss as a dominant-negative consequence of expression of the mutant protein. Allele-specific gene suppression by RNA interference (RNAi) is a potentially attractive strategy to prevent hearing loss caused by this mechanism. In this proof-of-principle study, we identified a potent GJB2-targeting short interfering RNA (siRNA) to post-transcriptionally silence the expression of the R75W allele variant of GJB2 in cultured mammalian cells. In a mouse model, this siRNA duplex selectively suppressed GJB2(R75W) expression by >70% of control levels, thereby preventing hearing loss. The level of endogenous murine Gjb2 expression was not affected. Our data show that RNAi can be used with specificity and efficiency in vivo to protect against hearing loss caused as a dominant-negative consequence of mutant gene expression.

Animals↗
PubMed2006

Delivery strategies for siRNA-mediated gene silencing.

RNA interference (RNAi) represents a promising new gene silencing technology for functional genomics and a potential therapeutic strategy for a variety of genetic diseases. RNAi involves the targeted post-transcriptional degradation of messenger RNA thereby inhibiting the synthesis of the desired protein. This effectively leads to silencing of gene expression. The effectors of this process are short interfering RNA (siRNA) duplexes (approximately 21-23nt) that are key intermediaries in the specific degradation of target mRNA following incorporation into the RNA-induced silencing complex (RISC) in the cytosol. However, due to the large molecular weight and negative charge of siRNA duplexes the effective cellular uptake and intracellular delivery appear to represent a major challenge for the widespread use of RNAi in vivo. This review summarises some of the main delivery strategies that have been attempted for the transfection of siRNA to cells in vitro and in vivo.

Administration, Intranasal↗
PubMed2006

Suppression of bovine viral diarrhea virus replication by small interfering RNA and short hairpin RNA-mediated RNA interference.

Bovine viral diarrhea virus (BVDV) is a ubiquitous viral pathogen that affects cattle herds' worldwide causing significant economic loss. The current strategies to control BVDV infection include vaccination (modified-live or killed) and control of virus spread by enhanced biosecurity management, however, the disease remains prevalent. With the discovery of the sequence-specific method of gene silencing known as RNA interference (RNAi), a new era in antiviral therapies has begun. Here we report the efficient inhibition of BVDV replication by small interfering (siRNA) and short hairpin RNA (shRNA)-mediated gene silencing. siRNAs were generated to target the 5' non-translated (NTR) region and the regions encoding the C, NS4B and NS5A proteins of the BVDV genome. The siRNAs were first validated using an EGFP/BVDV reporter system and were then shown to suppress BVDV-induced cytopathic effects and viral titers in cell culture with surprisingly different activities compared to the reporter system. Efficient viral suppression was then achieved by bovine 7SK-expressed BVDV-specific shRNAs. Overall, our results demonstrated the use of siRNA and shRNA-mediated gene silencing to achieve efficient inhibition of the replication of this virus in cell culture.

Animals↗
PubMed2003

Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat P2X3 receptor.

Synthetic 21-bp-long short interfering RNAs (siRNA) can stimulate sequence-specific mRNA degradation in mammalian cell cultures, a process referred to as RNA interference (RNAi). In the present study, the potential of RNAi was compared to the traditional antisense approach, acting mainly via RnaseH, for targeting the recombinant rat pain-related cation-channel P2X3 expressed in CHO-K1 and a rat brain tumour-derived cell line, 33B. Downregulation of the P2X3 receptor was evaluated at the mRNA, protein, and functional levels. In this study, four siRNA duplexes induced up to 95% sequence-specific inhibition of the P2X3 mRNA, independent of the type of 2 nt 3'-overhang modification and the location of the targeted sequences. Furthermore, we detected and characterised an independent combinatorial effect of antisense oligonucleotides (ASOs) and RNAi-mediated specific inhibition of the P2X3 receptor. Enhanced downregulation was observed only when siRNA was combined with nonhomologous ASO, targeting distant regions on the common P2X3 mRNA. The two reagents resulted in more efficient downregulation of P2X3 mRNA when administered in combination rather than separately. To our knowledge, this is the first investigation at the molecular level of the potential benefits of mixed antisense and RNAi-mediated treatment for inhibiting expression of a medically relevant pain-related gene.

Animals↗
PubMed2004

Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa.

RNA interference (RNAi) in animals, cosuppression in plants, and quelling in fungi are homology-dependent gene silencing mechanisms in which the introduction of either double-stranded RNA (dsRNA) or transgenes induces sequence-specific mRNA degradation. These phenomena share a common genetic and mechanistic basis. The accumulation of short interfering RNA (siRNA) molecules that guide sequence-specific mRNA degradation is a common feature in both silencing mechanisms, as is the component of the RNase complex involved in mRNA cleavage. During RNAi in animal cells, dsRNA is processed into siRNA by an RNase III enzyme called Dicer. Here we show that elimination of the activity of two Dicer-like genes by mutation in the fungus Neurospora crassa eliminates transgene-induced gene silencing (quelling) and the processing of dsRNA to an siRNA form. The two Dicer-like genes appear redundant because single mutants are quelling proficient. This first demonstration of the involvement of Dicer in gene silencing induced by transgenes supports a model by which a dsRNA produced by the activity of cellular RNA-dependent RNA polymerases on transgenic transcripts is an essential intermediate of silencing.

Base Sequence↗
PubMed2006

A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference.

RNA interference (RNAi) might be an efficient antiviral therapy for some obstinate illness. Herein, a retrovirus-based RNAi system was developed to drive expression and delivery of Hepatitis B virus (HBV)-specific short hairpin RNA (shRNA) in HepG2 cells. The levels of HBsAg and HBeAg and that of HBV mRNA were dramatically decreased by this RNAi system in HepG2 cells transfected with Topo-HBV plasmid. Retrovirus-based RNAi thus may be useful for therapy in HBV and other viral infections and provide new clues for prophylactic vaccine development.

Cell Line, Tumor↗
PubMed2002

Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi.

Eukaryotic heterochromatin is characterized by a high density of repeats and transposons, as well as by modified histones, and influences both gene expression and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, we deleted the argonaute, dicer, and RNA-dependent RNA polymerase gene homologs, which encode part of the machinery responsible for RNA interference (RNAi). Deletion results in the aberrant accumulation of complementary transcripts from centromeric heterochromatic repeats. This is accompanied by transcriptional de-repression of transgenes integrated at the centromere, loss of histone H3 lysine-9 methylation, and impairment of centromere function. We propose that double-stranded RNA arising from centromeric repeats targets formation and maintenance of heterochromatin through RNAi.

Centromere↗