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Results for “RNA Interference”

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At least 379 records · Page 21Linked to original sources

PubMed1999

RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles.

Animals↗

PubMed2004

Rational siRNA design for RNA interference.

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.

Animals↗

PubMed2006

Effects of RNA interference on CD80 and CD86 expression in bone marrow-derived murine dendritic cells.

To investigate whether RNA interference (RNAi) induced by small interfering RNA (siRNA) could suppress CD80 and CD86 expression in bone marrow-derived murine dendritic cells (DC). The bone marrow-derived DC of mice were separated and cultured in vitro, chemically synthesized siRNA were then transferred into the cells by LipofectAMINE 2000, and the siRNA transfection efficacy was assessed by both fluorescence microscope and flow cytometry. The mRNA expression and protein synthesis were analysed by real-time RT-PCR and flow cytometry. The cell viability of transfected DC was determined by annexin V and propidium iodine staining. Transfection of bone marrow-derived murine DC with a non-silencing FITC-labelled control siRNA demonstrated a high (71.86%) transfection efficiency without affecting cellular viability. CD80-1 siRNA was the most effective siRNA to block CD80 expression in three candidates. Similarly, CD86-3 siRNA was extraordinarily effective in repressing the expression of CD86. Cotransfection of siRNA specific to CD80 and CD86 can enhance gene silencing that is not affected by DC activation-inducing signals. CD80 and CD86 siRNA suppressed the expression of CD80 and CD86 to 31.05 +/- 2.41% and 25.43 +/- 0.85%, respectively, of the level in untreated cells (P < 0.05). siRNA is capable of triggering RNAi in bone marrow-derived DC; it can specifically and effectively knock down CD80 and CD86 gene expression. This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for allograft rejection.

Animals↗

PubMed2004

Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system.

RNA interference (RNAi) is a simple and powerful tool widely used for studying gene function in a number of species. Recently, inducible regulation of RNAi in mammalian cells using either tetracycline- or ecdysone-responsive systems has been developed to prevent potential lethality or non-physiological responses associated with persistent suppression of genes that are essential for cell survival or cell cycle progression. Here we show that the inducible regulation of RNAi also can be achieved by using a Cre-LoxP approach. We demonstrate that the insertion of a loxP-flanked neomycin cassette into RNA polymerase III promoter, which controls a vector-based RNAi unit, impairs the promoter activity. However, the expression of RNAi construct can be completely restored upon the removal of the neo cassette using a tamoxifen inducible Cre construct. We show that this system works with high efficiency in suppression of two endogenous genes, Fgfr2 and Survivin, in mouse embryonic stem (ES) cells, as evidenced by the decrease of levels of gene expression, reduced cell proliferation and colony formation. This system provides a potentially important yet simple approach to establish mutant mouse strains for functional study at defined stages upon turning on the inducible switches controlled by the Cre-LoxP system.

Animals↗

PubMed2003

Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin.

RNA interference is an effective method to silence specific gene expression. Its application to mammalian cells, however, has been hampered by various shortcomings. Recently, it was reported that introduction of 22-bp double-stranded RNAs (dsRNAs) would specifically suppress expression of endogenous and heterogeneous genes in various mammalian cell lines. However, using this method, we failed to knock out proteins of interest effectively. Here we report the development of a stable and controllable method for generating dsRNA intracellularly. Tetracycline-responsive transactivator-containing cells were transfected with a vector capable of tetracycline-induced bidirectionally overexpressing sense and antisense RNA to form dsRNA in vivo. With this method, glutaredoxin, monitored by Western blot, was knocked out by overexpressing 290-base sense and antisense RNA in NIH 3T3 cells controlled by tetracycline or doxycycline. By using these glutaredoxin knocked-out cells, we have demonstrated that actin deglutathionylation plays a key role in growth factor-mediated actin polymerization, translocalization, and reorganization near the cell periphery.

3T3 Cells↗

PubMed2000

A link between RNA interference and nonsense-mediated decay in Caenorhabditis elegans.

Double-stranded RNA (dsRNA) inhibits expression of homologous genes by a process involving messenger RNA degradation. To gain insight into the mechanism of degradation, we examined how RNA interference is affected by mutations in the smg genes, which are required for nonsense-mediated decay. For three of six smg genes tested, mutations resulted in animals that were initially silenced by dsRNA but then recovered; wild-type animals remained silenced. The levels of target messenger RNAs were restored during recovery, and RNA editing and degradation of the dsRNA were identical to those of the wild type. We suggest that persistence of RNA interference relies on a subset of smg genes.

Adenosine Deaminase↗

PubMed2004

CRE recombinase-inducible RNA interference mediated by lentiviral vectors.

Recently, several systems designed to trigger RNA interference by using small hairpin RNA driven by polymerase III promoters have been described. Here, we report a lentiviral-mediated small interfering RNA delivery system that can be induced by CRE recombinase. The system consists of a lentiviral vector carrying a mouse U6 promoter that is separated from a small hairpin RNA by a random DNA stuffer sequence flanked by modified loxP sites. The silencing cassette is not expressed until activated by addition of CRE recombinase delivered by a lentiviral vector. We have used this system to show specific down-regulation of GFP and two endogenous genes (the tumor suppressor p53 and the NF-kappaB transcription factor subunit p65) in vitro. Furthermore, down-regulation of both p53 and p65 resulted in the expected effect on downstream genes and cellular phenotype. We foresee multiple applications of this system both in vitro and in vivo to down-regulate specific targets in a tissue-specific and localized manner.

Base Sequence↗

PubMed2006

RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

Animals↗

PubMed2005

Transgene-induced RNA interference as a tool for plant functional genomics.

RNA interference (RNAi) is a powerful tool for functional genomics in a number of species. The logistics and procedures for doing high-throughput RNAi to investigate the functions of large numbers of genes in Arabidopsis thaliana and in Zea mays are described. Publicly available plasmid vectors that facilitate the stable chromosomal integration of inverted repeat transgenes that trigger RNAi have been used to generate more than 50 independent transgenic lines each in Arabidopsis and maize. Analysis of mRNA abundance of the targeted genes in independent lines transformed with distinct constructs indicates that the success of RNAi-induced silencing is gene dependent. mRNA levels were not detectably reduced for some genes, but were dramatically reduced for a number of genes targeted. A common pattern was that multiple independent lines transgenic for the same construct showed the same extent of silencing. This chapter describes the procedures used to generate and test transgenic lines mediating RNAi in Arabidopsis and maize.

Arabidopsis↗

PubMed2003

A simple "soaking method" for RNA interference in the planarian Dugesia japonica.

A simple method was developed for RNA interference (RNAi) in the planarian Dugesia japonica. The DjIFb ( Dugesia japonica intermediate filament b) gene was used to evaluate the effect of RNAi because both the cDNA and an antiserum against the gene product were available. After transverse cutting at the pre- and post-pharyngeal regions, the middle part of the body fragment was soaked in water containing double-stranded RNA (dsRNA) for about 5 h and then allowed to regenerate in water. On the 5th day of regeneration, little DjIFb protein was detected in the new tissues. When the worms were cut after soaking in dsRNA water, no RNAi effect was observed, suggesting that the dsRNA was introduced through the cut surface. A high concentration of dsRNA or repeated "cutting and soaking" resulted in more effective RNAi. This simple soaking method in combination with expressed sequence tag analysis should be very useful for high-throughput analyses of gene functions in planarian regeneration.

Animals↗

PubMed2006

RNA interference for antiviral therapy.

Silencing gene expression through a process known as RNA interference (RNAi) has been known in the plant world for many years. In recent years, knowledge of the prevalence of RNAi and the mechanism of gene silencing through RNAi has started to unfold. It is now believed that RNAi serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing RNAi have been found in various viral pathogens. During the past few years, it has been demonstrated that RNAi, induced by specifically designed double-stranded RNA (dsRNA) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. Furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. However, many key questions and obstacles in the translation of RNAi into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsRNA that induces the system. It is expected that for the specific examples in which the delivery issue could be circumvented or resolved, RNAi may hold promise for the development of gene-specific therapeutics.

Animals↗

PubMed2002

Specific and potent RNA interference in terminally differentiated myotubes.

Double-stranded RNA (dsRNA) interference is a potent mechanism for sequence-specific silencing of gene expression and represents an invaluable approach for investigating gene function in normal and diseased states as well as for drug target validation. Here, we report that skeletal muscle myoblasts and terminally differentiated myotubes are susceptible to RNA interference. We employed an approach in which dsRNA is generated by cellular transcription from plasmids containing long (1 kilobase) inverted DNA repeats of the target gene rather than using dsRNA synthesized in vitro. We show that gene silencing by this method is effective for endogenously expressed genes as well as for exogenous reporter genes. An analysis of the expression of several endogenous genes and exogenous reporters demonstrates that the silencing effect is specific for the target gene containing sequences within the inverted repeat. Our method eliminates the need to chemically synthesize dsRNA and is not accompanied by global repression of gene expression. Furthermore, we show for the first time that sequence-specific dsRNA-mediated gene silencing is possible in differentiated, multinucleated skeletal muscle myotubes. These findings provide an important molecular tool for the examination of protein function in terminally differentiated muscle cells and provide alternative approaches for generating disease models.

Animals↗

PubMed2005

The genesis of RNA interference, its potential clinical applications, and implications in gynecologic cancer.

OBJECTIVE: This review will discuss the discovery and development of RNA interference (RNAi) technology, small interfering RNA (siRNA) design and delivery, and the implications of RNAi on gynecologic cancers. METHODS: Systematic review of English language literature using searches for RNAi and gynecologic cancers in established databases, including Pubmed and Ovid, was employed. RESULTS: The high degrees of efficiency and specificity are the main advantages of RNAi. Consequently, RNAi is used in functional genomics and developing therapies for the treatment of viral infection, dominant disorders, neurological disorders, and cancers, including gynecologic cancers. CONCLUSION: RNAi represents an exciting technology for functional genomics by selective targeting of genes. While issues regarding delivery remain, the therapeutic advantages of siRNA in cancer treatment warrant further investigation.

Animals↗

PubMed2003

RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells.

The human papillomavirus oncoproteins E6 and E7 promote cell proliferation and contribute to carcinogenesis by interfering with the activities of cellular tumor suppressors. We used a small interfering RNA molecule targeting the E7 region of the bicistronic E6 and E7 mRNA to induce RNA interference, thereby reducing expression of E6 and E7 in HeLa cells. RNA interference of E6 and E7 also inhibited cellular DNA synthesis and induced morphological and biochemical changes characteristic of cellular senescence. These results demonstrate that reducing E6 and E7 expression is sufficient to cause HeLa cells to become senescent.

Cell Transformation, Viral↗

PubMed2005

Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.

Animals↗

PubMed2005

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.

RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.

Alternative Splicing↗

PubMed2006

Gene silencing through RNA interference (RNAi) in vivo: strategies based on the direct application of siRNAs.

RNA interference (RNAi) offers great potential not only for in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene, e.g. in tumor therapy. Since it relies on small interfering RNAs (siRNAs), which are the mediators of RNAi-induced specific mRNA degradation, a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on (viral) vector delivery may be of only limited clinical use. The more desirable approach is to directly apply catalytically active siRNAs. This review highlights the recent knowledge on the guidelines for the selection of siRNAs which show high activity in the absence of non-specific siRNA effects. It then focuses on approaches to directly use siRNA molecules in vivo and gives a comprehensive overview of in vivo studies based on the direct application of siRNAs to induce RNAi. One promising approach is the in vivo siRNA delivery through complexation of chemically unmodified siRNAs with polyethylenimine (PEI). The anti-tumoral effects of PEI/siRNA-based targeting of tumor-relevant genes in vivo are described.

Gene Silencing↗

PubMed2005

[Down-regulation of CT120A by RNA interference suppresses lung cancer cells growth].

OBJECTIVE: To validate our obtained outcomes and clarify the relationship between CT120A, a novel human plasma membrane-associated gene, and proliferation of lung cancer cells. METHODS: A vector-based small hairpin RNA (shRNA) was transfected into the human lung adenocarcinoma SPC-A-1 cells to specifically target CT120A cDNA. RT-PCR and Western blotting were used to analyze the CT120A expression. The cell proliferation rate was analyzed by BrdU-TdR incorporation assay, the ability of cells to grow in soft agarose and the tumorigenicity in nude mice were measured. Flow cytometry was performed to analyze cell apoptosis. RESULTS: When compared with the scrambled control cell line, CT120A transcripts were reduced by 70% and 50% in two shRNA-H stable transfectants, H2 and H3 clones, respectively. The protein of CT120A was reduced by about 80% in both the H2 and H3 clones. By BrdU incorporation assay, up to the 6th day a dramatic decrease in the cell growth rate (30% to 40%) was observed in the shRNA-H2 and shRNA-H3 cell lines. The colony formation rate in soft agarose of the two cell lines was about one half that of the control cells. In addition, a remarkable reduction of tumorigenicity of the two cell lines was observed as compared with that of the control. The suppression of CT120A expression also sensitized cells to ultraviolet-induced apoptosis. CONCLUSION: Down-regulation of CT120A by RNA interference suppresses lung cancer cell growth. The successful knockdown of CT120A expression by RNA interference implicates that CT120A may be a new candidate of drug target for treatment of lung cancers.

Adenocarcinoma↗