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At least 127 records · Page 7Linked to original sources
PubMed2004

Inducible, reversible, and stable RNA interference in mammalian cells.

RNA interference is a powerful genetic approach for efficiently silencing target genes. The existing method of gene suppression by the constitutive expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes but limits the analysis of genes essential for cell survival, cell cycle regulation, and cell development. We have developed an inducible U6 promoter for synthesis of shRNAs in both human and murine cells. Cells containing stably integrated shRNA expression constructs demonstrate stringent dosage- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer ecdysone. Inducible suppression of human p53 in glioblastoma cells shows striking morphological changes and defects in cell cycle arrest caused by DNA damage, as expected. Remarkably, the inducibility is reversible after withdrawal of the inducer, as observed by reappearance of the protein and a restoration of the original cell phenotype. Inducible and reversible regulation of RNA interference has broad applications in the areas of mammalian genetics and molecular therapeutics.

Animals↗
PubMed2003

RNA interference in mammalian cells by chemically-modified RNA.

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.

Blotting, Western↗
PubMed2005

A member of the polymerase beta nucleotidyltransferase superfamily is required for RNA interference in C. elegans.

RNA interference (RNAi) is an ancient, highly conserved mechanism in which small RNA molecules (siRNAs) guide the sequence-specific silencing of gene expression . Several silencing machinery protein components have been identified, including helicases, RNase-related proteins, double- and single-stranded RNA binding proteins, and RNA-dependent RNA polymerase-related proteins . Work on these factors has led to the revelation that RNAi mechanisms intersect with cellular pathways required for development and fertility . Despite rapid progress in understanding key steps in the RNAi pathway, it is clear that many factors required for both RNAi and related developmental mechanisms have not yet been identified. Here, we report the characterization of the C. elegans gene rde-3. Genetic analysis of presumptive null alleles indicates that rde-3 is required for siRNA accumulation and for efficient RNAi in all tissues, and it is essential for fertility and viability at high temperatures. RDE-3 contains conserved domains found in the polymerase beta nucleotidyltransferase superfamily, which includes conventional poly(A) polymerases, 2'-5' oligoadenylate synthetase (OAS), and yeast Trf4p . These findings implicate a new enzymatic modality in RNAi and suggest possible models for the role of RDE-3 in the RNAi mechanism.

Amino Acid Sequence↗
PubMed2006

[Knockdown of Hsp701A induces K562 cells apoptosis by RNA interference].

AIM: To investigate the apoptosis of K562 cells induced by RNA interference(RNAi) targeting Hsp701A. METHODS: Small interference RNA (siRNA) targeting Hsp701A was generated and its eukaryotic expression vectors was constructed and transected into K562 cells. Proliferation, apoptosis and cell cycle of the transected cells was respectively detected by MTT colorimetry and FCM. RESULTS: The eukaryotic expression vector of siRNA against Hsp701A was successfully constructed and transfected into K562 cells, which markedly decreased the expression of Hsp701A on both mRNA and protein level. K562 cells transfected with the vector exhibited slower proliferation, increased apoptosis and increased G(0)/G(1) arrest. CONCLUSION: The inhibition of Hsp701A gene by RNAi may provide a foundation for the development of novel therapeutic strategy for chronic granulocyte leukemia (CML).

Apoptosis↗
PubMed2006

Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter.

RNA interference (RNAi) mediated by expression of short hairpin RNAs (shRNAs) is a powerful tool for efficiently suppressing target genes. The approach allows studies of the function of individual genes and may also be applied to human therapy. However, in many instances regulation of RNAi by administration of a small inducer molecule will be required. To date, the development of appropriate regulatory systems has been hampered by the few possibilities for modification within RNA polymerase III promoters capable of driving efficient expression of shRNAs. We have developed an inducible minimal RNA polymerase III promoter that is activated by a novel recombinant transactivator in the presence of doxycycline (Dox). The recombinant transactivator and the engineered promoter together form a system permitting regulation of RNAi by Dox-induced expression of shRNAs. Regulated RNAi was mediated by one single lentiviral vector, blocked the expression of green fluorescent protein (GFP) in a GFP-expressing HEK 293T derived cell line and suppressed endogenous p53 in wild-type HEK 293T, MCF-7 and A549 cells. RNA interference was induced in a dose- and time-dependent manner by administration of Dox, silenced the expression of both target genes by 90% and was in particular reversible after withdrawal of Dox.

Cell Line↗
PubMed2002

RNA interference in adult mice.

RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

Aging↗
PubMed2005

Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes.

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.

Animals↗
PubMed2005

Novel methods for expressing RNA interference in human cells.

RNA interference (RNAi) is a conserved process in which a double-stranded RNA (dsRNA) induces sequence-specific gene silencing. Recent developments in the use of the 21-nt small interfering RNA (siRNA) have allowed the specific degradation of mRNA without induction of nonspecific effects in mammalian cells. RNAi provides a method for knocking down genes of interest and a powerful tool for studies on gene functions in various organisms. Although many vector-based siRNA expression systems have been developed for production of siRNAs in mammalian cells, many technical issues for an effective production of siRNAs still need to be resolved. In this chapter, we describe methods for construction of genetically stable and highly active siRNA expression systems and also mention some strategies to overcome serious technical problems.

Base Sequence↗
PubMed2004

Genes required for systemic RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) in the nematode worm, Caenorhabditis elegans, occurs systemically. Double-stranded RNA (dsRNA) provided in the diet can be absorbed from the gut lumen and distributed throughout the body, triggering RNAi in tissues that are not exposed to the initial dsRNA trigger. This is in marked contrast to other animals, in which RNAi does not spread from targeted tissues to neighboring cells. Here, we report the characterization of mutants defective in the systemic aspect of RNAi, but not in the core RNAi process itself. Analysis of these mutants suggests that dsRNA uptake is a specific process involving several unique proteins.

Amino Acid Sequence↗
PubMed2006

RNA interference and potential applications.

RNA interference (RNAi) is the process of using specific sequences of double-stranded RNA (dsRNA) to knock down the expression level of sequence-homologous genes. Such ability of small interfering RNA (siRNA) in mammalian cells will undoubtedly revolutionize the study of functional genomics, the discovery of drug targets and even the treatment of human diseases. In this review we briefly describe the history of RNAi discovery, the RNAi mechanism and the general guideline for siRNA design as well as various methods for siRNA production and delivery. We also introduce the potential applications of siRNA, inducible siRNA and siRNA library in speeding up basic biomedical research and in acting as potential therapeutic agents for treatment of numerous human diseases.

Animals↗
PubMed2006

ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC) transporter gene superfamily. ABC transporters use ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells, organellar biogenesis, and mechanisms that protect against viral infection. HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that eight additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that use ABC proteins.

ATP-Binding Cassette Transporters↗
PubMed2006

High-throughput RNA interference in functional genomics.

RNA interference (RNAi) refers to post-transcriptional silencing of gene expression as a result of the introduction of double-stranded RNA into cells. The application of RNAi in experimental systems has significantly accelerated elucidation of gene functions. In order to facilitate large-scale functional genomics studies using RNAi, several high-throughput approaches have been developed based on microarray or microwell assays. The recent establishment of large libraries of RNAi reagents combined with a variety of detection assays has further improved the performance of functional genome-wide screens in mammalian cells.

Animals↗
PubMed2005

Labeling and characterization of small RNAs associated with the RNA interference effector complex RITS.

RNA interference (RNAi) is a gene silencing mechanism that acts at both the posttranscriptional and transcriptional levels. We have recently identified an RNA-containing complex, named RNA-induced transcriptional silencing (RITS), that directly links RNAi to transcriptional gene silencing in Schizosaccharomyces pombe. Here we review the affinity purification methods we use to isolate RITS and describe how to purify, detect, and analyze RNAs associated with this complex.

Blotting, Southern↗
PubMedDate not supplied

RNA interference may be more potent than antisense RNA in human cancer cell lines.

1. RNA interference (RNAi) is a newly discovered cellular pathway for the silencing of sequence-specific genes at the mRNA level by the introduction of the cognate double-stranded (ds) RNA. Because antisense (AS) mechanisms have similar effects, we compared these two effects in human cancer cell lines, considering a possible application of RNAi for cancer therapy. 2. We tested RNAi effects by transfecting human hepatoma and pancreatic cancer cell lines with AS and sense (S) RNA expression plasmids corresponding to the exogenous luciferase gene or the endogenous c-raf gene in the form of complexes with a cationic lipopolyamine or a tumour-targeting peptide vector we developed. In addition, we compared the effects of small interfering RNA and AS oligoDNA complexed with the peptide vector. 3. From the viewpoint of AS actions, the effect of the AS RNA may be cancelled by the S RNA, although, interestingly, we found that the combination of the AS and S RNA expression plasmids was more effective than the AS RNA expression plasmids alone in reducing target gene expression, whereas the S RNA expression plasmids had no effects. The combination of the luciferase AS and S RNA had no effects on the expression of either the beta-galactosidase gene or the c-raf gene. In the presence of 2-aminopurine (an inhibitor of dsRNA-activated protein kinase), the inhibitory effect of the combination of AS and S RNA on gene expression did not change in the case of the endogenous c-raf gene, but was reduced in the case of the exogenous luciferase gene. The effect of 22 nucleotide RNA duplexes corresponding to the luciferase gene was by one order stronger than that of the phosphorothioate AS DNA. 4. Thus, it is suggested that RNAi may be more potent than AS RNA in reducing target gene expression in human cancer cell lines, regardless of the length of dsRNA. With further studies on the RNAi phenomenon in cancer cells, RNAi could provide a novel approach for cancer gene therapy.

2-Aminopurine↗
PubMed2003

Selective inhibition of hepatitis B virus replication by RNA interference.

Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively.

Animals↗
PubMed2004

Towards therapy using RNA interference.

Small interfering RNA (siRNA) molecules are short sequences of double-stranded RNA 19-27 bp in length, which suppress expression of target genes by inducing the breakdown of the cognate mRNA through mechanisms that are still being elucidated. siRNA molecules can be chemically synthesized or prepared through digestion of larger double-stranded RNA molecules using recombinant dicer or RNAase III enzyme. siRNA molecules can also be encoded by plasmid or virus vectors or expressed in transgenic animals. Design of siRNA sequences that efficiently suppress target genes can sometimes be challenging, although digestion of large double-stranded RNA species with recombinant dicer or RNAase III may remove the necessity for testing multiple candidate siRNA. Exogenous siRNA can suppress translation for varying amounts of time depending on the half-life of the protein targeted. Vector-mediated approaches may improve duration but their use can be limited by the permanency and efficiency of transduction. Potential therapeutic targets for siRNA include viral and non-viral pathogens, cancer, neurodegenerative diseases, septic shock and macular degeneration. Suppression of expression via siRNA is also an extremely useful research tool for ascertaining gene function. Looking ahead to clinical applications, it will be important to know the consequences of inadvertent suppression of non-targeted sequences. If safety can be established, siRNA has the potential to significantly impact the field of molecular medicine.

Clinical Trials as Topic↗
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↗
PubMed2005

Disruption of GW bodies impairs mammalian RNA interference.

The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs). GWBs are enriched in proteins that are involved in mRNA degradation. Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures. In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing.

Argonaute Proteins↗