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Results for “RNA Interference”

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At least 19 recordsLinked to original sources
PubMed2002

RNA interference (RNA(i)) induction with various types of synthetic oligonucleotide duplexes in cultured human cells.

Various types of synthetic oligonucleotide duplexes against the Photinus luciferase gene were tested on their induction of the sequence-specific RNA interference (RNAi) activity in transfected human cells. Results indicate that RNA duplexes with ribonucleotide 3' overhangs rather than those with deoxyribonucleotide 3' overhangs induce more efficient RNAi activity, and that sense-stranded DNA/antisense-stranded RNA hybrids induce a moderate RNAi activity. These results suggest that there is a difference in the potential of oligonucleotide duplexes to be RNAi mediators, i.e. short interfering RNAs (siRNAs), between human RNAi and invertebrate RNAi. The data further show that different siRNAs induce different levels of RNAi.

Animals↗
PubMed2005

Selective sensitivity of Caenorhabditis elegans neurons to RNA interference.

RNA interference is a new approach to knockdown gene expression, but effectiveness varies depending on the organism, cell type or target sequence. Studies with Caenorhabditis elegans have shown that subsets of cells including neurons are often resistant to RNA interference. We measured RNA interference using green fluorescent protein reporter strains and feeding, soaking and injection delivery methods in a number of Caenorhabditis elegans neuron subtypes (dopaminergic, GABAergic, cholinergic, glutamatergic, touch). The sensitivity to RNA interference varied: GABAergic and dopaminergic neurons showed greater resistance while cholinergic, glutamatergic and touch neurons were more sensitive. Dysfunctional RRF-3, a putative RNA-directed RNA polymerase, had a significant effect on increasing neuron sensitivity in most subtypes. These results demonstrate that Caenorhabditis elegans neurons vary in their sensitivity to RNA interference.

Acetylcholine↗
PubMed2005

Inhibition of drug-resistant HIV-1 by RNA interference.

RNA interference is a powerful tool used to inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. Almost all HIV-1 genes have been targets for small interfering RNA (siRNA) molecules, and HIV-1 replication can be specifically and successfully inhibited by this technique. RNA interference has been proposed as an alternative strategy to inhibit replication of drug-resistant viruses that emerge during suboptimal antiretroviral therapy for HIV-1. To investigate specific inhibition of drug-resistant HIV-1 by RNA interference, we designed siRNA molecules that recognize codons 181-188 of the reverse transcriptase (RT) gene of wild-type HIV-1 and HIV-1 carrying the M184V mutation, which confers high-level resistance to the RT inhibitor lamivudine. Using viral variants with single point mutations at codon 184, we measured the impact of these mutations on virus replication. We have demonstrated that siRNA targeting either wild-type HIV-1 or M184V variants inhibits replication of the corresponding virus, but does not influence replication of virus with a mismatch in the targeted region. Combining two effective siRNAs did not show synergistic inhibitory effect on HIV-1 replication. However, a combination of lamivudine and siRNA-M184V was very effective in inhibiting replication of both wild-type and variant M184V viruses in mixed infection experiments. Taken together, these results demonstrate that RNA interference might be useful in the treatment of drug-resistant HIV-1 infection.

Dose-Response Relationship, Drug↗
PubMed2004

Pharmaceutical prospects for RNA interference.

RNA interference has proven to be a powerful tool in gene function validation. Recently, the first studies were published reporting a disease-modulating activity of the technique, suggesting a promise for RNA interference as a novel therapeutic strategy. This review discusses the recent advancements in realizing the clinical utility of RNA-interference.

Animals↗
PubMed2006

Gene silencing in adult rat cardiac myocytes in vitro by adenovirus-mediated RNA interference.

RNA interference (RNAi) by short double stranded RNA (siRNA) represents an efficient and frequently used tool for gene silencing to study gene function. Whereas efficient ablation of genes has been demonstrated in neonatal cardiac myocytes, thus far information on successful application of this technique in adult cardiac myocytes (ACM), a standard experimental model in cardiac physiology and pathophysiology, is sparse. Here we demonstrate efficient ablation of a transgene encoding for enhanced green fluorescent protein (EGFP) and a cell specific endogenous gene encoding for an inward-rectifier channel subunit (Kir2.1) in ACM in vitro using adenovirus driven transcription of siRNA hairpins. EGFP fluorescence and density of background inward rectifier current (IK1) were reduced by > 90% within about 6-8 days after transformation with the corresponding virus. In Kir2.1-silenced myocytes resting membrane potential was significantly reduced. Survival of these cells in culture was compromised, presumably due to Ca2+ -overload caused by the depolarization. The sequence-specific knockdowns of EGFP and Kir2.1 were confirmed on the RNA level using real-time RT-PCR. In Kir2.1-silenced myocytes density of transient outward current, carried predominantly by Kv4.x subunits remained unaffected. This communication for the first time demonstrates proof of principle of efficient RNA interference using adenovirus-based vectors and demonstrates its large potential in phenotyping of ACM.

Adenoviridae↗
PubMedDate not supplied

[Vector systems of RNA interference].

RNA interference is a mechanism of posttranslational (at the level of mRNA) gene silencing. Sequence-specific mRNA degradation is realized with the help of small interfering RNAs produced by processing of a precursor using Dicer, an enzyme from the RNAse III family. This mechanism is now widely used in vitro on cultures of mammalian cells in order to elucidate functions of individual genes by gene specific knockdown. Analogs of small interference RNAs are intensely expressed during embryogenesis. The mechanism of RNA interference plays an especially important role in embryogenesis of invertebrates. Identification of the functions of small noncoding RNAs is essential for understanding the genetic mechanisms underlying individual developmental stages. In order to integrate small interference RNAs in mammalian cells, various systems have been developed that allow both transient (for 48 h) and stable expression in vitro. These systems are considered in the present review.

Animals↗
PubMed2004

Genes required for Drosophila nervous system development identified by RNA interference.

RNA interference was used to screen 3,314 Drosophila double-stranded RNAs, corresponding to approximately 25% of Drosophila genes, for genes that affect the development of the embryonic nervous system. RNA-interference-mediated gene silencing in Drosophila embryos resulted in loss-of-function mutant phenotypes for 43 genes, which is 1.3% of the genes that were screened. We found 18 genes that were not known previously to affect the development of the nervous system. The functions of some of the genes are unknown. Other genes encode protein kinases, transcription factors, and signaling proteins, as well as proteins with other functions.

Animals↗
PubMed2003

Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference.

RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a lentivirus vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As lentivirus vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.

Cell Line↗
PubMed2004

Inhibition of virus production in JC virus-infected cells by postinfection RNA interference.

RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.

Capsid Proteins↗
PubMed2006

Innate immune defense through RNA interference.

RNA interference (RNAi, also known as RNA silencing) has recently emerged as a fundamental and widespread regulator of gene expression. New developments in this field implicate RNAi in the innate immune response to infection in plants and animals. Evidence from plants, tissue culture cells, and Caenorhabditis elegans-based systems previously suggested that RNAi plays a role in the defense against viral infection, but definitive evidence using viruses and whole animals has been lacking. Two recent reports now show that both Drosophila embryos and adult flies mount a substantial innate immune response to insect viruses that requires the RNAi machinery. This innate response is distinct from known bacterial and fungal defense systems provided by the Toll and immune deficiency (Imd) pathways, thus defining a previously unrecognized strategy to fight viral infection. Whether RNAi, aside from its function in counteracting viruses, is also used to fight bacterial infection remained enigmatic. New evidence, however, now shows that in Arabidopsis, the bacterial component, flagellin, induces the expression of a specific microRNA, which in turn leads to the down-regulation of the signaling pathways that are implicated in disease susceptibility. This down-regulation then increases the plant's resistance to infection. Whether RNAi mechanisms also exist for combating bacterial diseases in animals remains an intriguing question for future studies.

Animals↗
PubMed2005

Functional genomics using high-throughput RNA interference.

RNA interference (RNAi) describes the post-transcriptional silencing of gene expression that occurs in response to the introduction of double-stranded RNA into cells. Application of RNAi in experimental systems has provided a great leap forward in the elucidation of gene function. To facilitate large-scale functional genomics studies using RNAi, several high throughput approaches have been developed based on microarray or microwell assays. Recent establishment of large libraries of RNAi reagents combined with a variety of detection assays further opens the door for genome-wide screens of gene function in mammalian cells.

Animals↗
PubMed2004

Gene silencing of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis by RNA interference.

RNA interference (RNAi) has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned Haemaphysalis longicornis serine proteinase (HlSP) gene to test the importance of the function of the HlSP gene product during blood-feeding. Growth of unfed ticks treated with HlSP dsRNA was significantly inhibited compared to that of PBS-treated ticks. This inhibition was supported by the level of HlSP mRNA. HlSP may play a crucial role for blood-feeding in these ticks. This is the first report on gene silencing of a functional serine proteinase in hard ticks.

Animals↗
PubMed2005

A computational study of off-target effects of RNA interference.

RNA interference (RNAi) is an intracellular mechanism for post-transcriptional gene silencing that is frequently used to study gene function. RNAi is initiated by short interfering RNA (siRNA) of approximately 21 nt in length, either generated from the double-stranded RNA (dsRNA) by using the enzyme Dicer or introduced experimentally. Following association with an RNAi silencing complex, siRNA targets mRNA transcripts that have sequence identity for destruction. A phenotype resulting from this knockdown of expression may inform about the function of the targeted gene. However, 'off-target effects' compromise the specificity of RNAi if sequence identity between siRNA and random mRNA transcripts causes RNAi to knockdown expression of non-targeted genes. The complete off-target effects must be investigated systematically on each gene in a genome by adjusting a group of parameters, which is too expensive to conduct experimentally and motivates a study in silico. This computational study examined the potential for off-target effects of RNAi, employing the genome and transcriptome sequence data of Homo sapiens, Caenorhabditis elegans and Schizosaccharomyces pombe. The chance for RNAi off-target effects proved considerable, ranging from 5 to 80% for each of the organisms, when using as parameter the exact identity between any possible siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived from a dsRNA (range 100-400 nt) representing the coding sequences of target genes and all other siRNAs within the genome. Remarkably, high-sequence specificity and low probability for off-target reactivity were optimally balanced for siRNA of 21 nt, the length observed mostly in vivo. The chance for off-target RNAi increased (although not always significantly) with greater length of the initial dsRNA sequence, inclusion into the analysis of available untranslated region sequences and allowing for mismatches between siRNA and target sequences. siRNA sequences from within 100 nt of the 5' termini of coding sequences had low chances for off-target reactivity. This may be owing to coding constraints for signal peptide-encoding regions of genes relative to regions that encode for mature proteins. Off-target distribution varied along the chromosomes of C.elegans, apparently owing to the use of more unique sequences in gene-dense regions. Finally, biological and thermodynamical descriptors of effective siRNA reduced the number of potential siRNAs compared with those identified by sequence identity alone, but off-target RNAi remained likely, with an off-target error rate of approximately 10%. These results also suggest a direction for future in vivo studies that could both help in calibrating true off-target rates in living organisms and also in contributing evidence toward the debate of whether siRNA efficacy is correlated with, or independent of, the target molecule. In summary, off-target effects present a real but not prohibitive concern that should be considered for RNAi experiments.

Animals↗
PubMed2004

Adenovirus vector-mediated doxycycline-inducible RNA interference.

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.

Adenoviridae↗
PubMed2003

Interfering with disease: opportunities and roadblocks to harnessing RNA interference.

RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. Short double-stranded RNAs, known as small interfering RNAs (siRNA), are incorporated into an RNA-induced silencing complex that directs degradation of RNA containing a homologous sequence. RNAi has been shown to work in mammalian cells, and can inhibit viral infection and control tumor cell growth in vitro. Recently, it has been shown that intravenous injection of siRNA or of plasmids expressing sequences processed to siRNA can protect mice from autoimmune and viral hepatitis. RNAi could provide an exciting new therapeutic modality for treating infection, cancer, neurodegenerative disease and other illnesses.

Animals↗
PubMed2003

Promoter choice affects the potency of HIV-1 specific RNA interference.

RNA interference (RNAi) is mediated by small interfering (si) RNAs that target and degrade mRNA in a sequence-specific manner. Cellular expression of siRNA can be achieved by the use of expression cassettes driven by RNA polymerase III (pol III) promoters. Here, we demonstrate that a modified tRNA(met)-derived (MTD) promoter effectively drives the cellular expression of HIV-1-specific siRNA. We observed up to 56% greater inhibition of virus production when the MTD promoter was used to drive the expression of short hairpin (sh) RNA targeting the HIV-1 transactivator protein tat compared to cassettes containing other pol III promoters such as H1, U6+1 and U6+27. We conclude that the MTD promoter is ideally suited to drive intracellular expression of HIV-1 specific siRNA and may serve as an important component of future RNAi vector delivery systems.

Cell Line↗
PubMed2006

In vivo and in vitro knockdown of FREP2 gene expression in the snail Biomphalaria glabrata using RNA interference.

RNA interference (RNAi) is reported here for the first time for Biomphalaria glabrata, the snail intermediate host for the human parasite Schistosoma mansoni. The fibrinogen-related protein 2 (FREP2) gene, normally expressed at increased levels following exposure to digenetic trematode parasites, such as S. mansoni or Echinostoma paraensei, was targeted for knockdown. Double-stranded RNA (dsRNA) corresponding to specific regions of the FREP2 gene was introduced into snails by direct injection into the hemolymph, 2 days prior to exposure to trematodes, or added to co-cultures of B. glabrata embryonic (Bge) cells and E. paraensei sporocysts. After introduction of FREP2 dsRNA, expression levels of FREP2 were significantly reduced, to 20-30% of control values. In addition, we were able to disrupt expression of the house-keeping myoglobin gene, further confirming the feasibility of RNAi for B. glabrata. Cross-reactivity in RNAi has not been observed either among four FREP gene subfamilies or between FREP2 and myoglobin. Establishment of RNAi techniques in B. glabrata provides an important tool for clarifying the function of genes believed to play a role in host-parasite interactions, specifically between B. glabrata and its trematode parasites, including S. mansoni.

Animals↗
PubMed2004

Technique review: how to use RNA interference.

RNA interference (RNAi) has been rapidly adopted as a general method for inhibiting gene expression in most laboratory organisms. This paper discusses how libraries of RNAi reagents are being used to perform genome-wide reverse genetic screens in both model organisms and mammalian cells. Guidelines for designing effective small interfering RNAs and appropriate controls for mammalian RNAi experiments will also be discussed.

Animals↗