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Results for “RNA interference”

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At least 1,063 records · Page 59Linked to original sources
PubMed2001

Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells.

Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

Animals↗
PubMed2005

RNA interference of Xenopus NMDAR NR1 in vitro and in vivo.

Here, we present a short interfering RNA (siRNA) application that reduces the expression of NR1 the obligate subunit of the NMDA receptor (NMDAR) and virtually eliminates NMDAR function in a small subset of neurons within otherwise normally developing Xenopus laevis tadpoles. We designed two plasmids each containing a CMV promoter driving a dsRed "reporter" cDNA and DNA coding for one short hairpin RNA (shRNA) under the control of the U6 promoter. The shRNA was cleaved to produce a siRNA against NR1 transcript (iNR1). NR1 transcript and protein, differentiation and survival of NR1 knockdown neurons, were assayed in vivo. iNR1 effects on NMDAR function used Xenopus tectal neuron cultures, Ca2+ imaging and patch-clamp electrophysiology. NR1 transcript, protein and NMDAR function was significantly reduced or eliminated in iNR1-expressing neurons. Protein, transcript and function for the closely related AMPA receptor (AMPAR) subunit GluR1 was unperturbed. In vivo imaging of small groups of iNR1 neurons at two different ages revealed that siRNA knockdown of the NMDAR does not affect differentiation or survival of young neurons. Thus, with siRNA, molecular hypotheses about synaptic mechanisms of circuit formation can be rapidly tested in intact developing vertebrates using normal neurons in the same animals as controls.

Animals↗
PubMed2002

RNA interference of peroxisome-related genes in C. elegans: a new model for human peroxisomal disorders.

RNA-mediated interference (RNAi) for the posttranscriptional silencing of genes was used to evaluate the importance of various peroxisomal enzymes and peroxins for the development of Caenorhabditis elegans and to compare the roles of these proteins in the nematode to their roles in yeasts and humans. The nematode counterparts of the human ATP-binding cassette half-transporters, the enzymes alkyldihydroxyacetonephosphate synthase and Delta(3,5)-Delta (2,4)-dienoyl-CoA isomerase, the receptors for peroxisomal membrane and matrix proteins (Pex19p and Pex5p), and components of the docking and translocation machineries for matrix proteins (Pex13p and Pex12p) are essential for the development of C. elegans. Unexpectedly, RNAi silencing of the acyl-CoA synthetase-mediated activation of fatty acids, the alpha- and beta-oxidation of fatty acids, the intraperoxisomal decomposition of hydrogen peroxide, and the peroxins Pex1p, Pex2p, and Pex6p had no apparent effect on C. elegans development. The described analysis of functional gene knockouts through RNAi provides a basis for the use of C. elegans as a valuable model system with which to study the molecular and physiological defects underlying the human peroxisomal disorders.

ATP-Binding Cassette Transporters↗
PubMed2005

RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development.

Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference-based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions.

Arabidopsis Proteins↗
PubMed2002

[Double stranded RNA (RNA interference RNAi) in different creatures].

Double stranded RNA could degrade mRNA of target gene. It is a useful way for studying gene function. It is used widely in different creatures,such as Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Zebrafish, Mouse. This review is mainly related to the application of dsRNA in recent years.

English Abstract↗
PubMed2000

Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating "knock-out" phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.

Animals↗
PubMed2005

[Therapeutic effects of RNA interference targeting HIF-1 alpha gene on human osteosarcoma].

OBJECTIVE: To investigate the inhibition effect of the small hairpin RNA (shRNA) targeting HIF-1alpha gene on the growth of osteosarcoma in vitro and in vivo. METHODS: The small hairpin RNA (shRNA) eukaryotic expression vector targeting HIF-1alpha gene, named pSilencer-HIF, was constructed and transfected into cultured human osteosarcoma cell of line SaOS-2 via liposome reagent. Then the osteosarcoma cells were cultured under chemical hypoxia conditions. The inhibition effects on HIF-1alpha gene were determined by semi-quantitative reverse transcription PCR and Western blot analysis. The in vitro cellular growth activities were assayed by MTT colorimetry. The cell apoptosis was studied by electron microscopy, TUNEL assay, and annexin V/PI double staining. Eighteen Balb/C mice were randomly divided into 3 equal groups to be inoculated with SaOS-2/shRNA, SaOS-2/neo (blank vector), or SaOS-2 subcutaneously respectively and then the appearance and size of tumors were observed. Four weeks later the mice were killed and the volumes of tumor were calculated so as to evaluate the therapeutic effects of shRNA. RESULTS: The successful construction of pSilencer-HIF plasmid was identified with sequencing. After the shRNA expression vector was transfected into the SaOS-2 cells, the expression of HIF-1alpha gene was inhibited significantly (by 90%). The cellular growth activities in the SaOS-2 cells transfected with pSilencer-HIF plasmid decreased obviously in hypoxia culture. After 72 hours of exposure to hypoxia, electron microscopy and TUNEL assay showed classic apoptosis characters in the SaOS-2 cells transfected with pSilencer-HIF plasmid with an apoptosis rate of 18.71% +/- 0.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the nontransfected group (both P < 0.01). The growth speed and formation rate of xenograft tumor in pSilencer-HIF transfected mice slowed down significantly. A lot of necrotic tissues could be observed in the pSilencer-HIF transfected group by HE staining, however, there was no similar inhibitive effect in the control groups. CONCLUSION: shRNA targeting HIF-1alpha gene blocks the hypoxia transduction pathway efficiently and inhibits the growth of osteosarcoma cells.

Animals↗
PubMed2005

Analysis of ARD1 function in hypoxia response using retroviral RNA interference.

Cellular hypoxia response is regulated at the level of hypoxia-inducible factor (HIF) activity. A number of recently identified oxygen sensors are HIF-modifying enzymes that respond to low oxygen by altering HIF modification and thus lead to its activation. In addition to the HIF proline hydroxylases and asparagine hydroxylases, ARD1 is recently described as a HIF-1alpha acetylase that regulates its stability. We found that ARD1 is down-regulated in a number of cell lines in response to hypoxia and hypoxia mimic compounds. After surveying these lines for erythropoietin production and retroviral transfection efficiency, we chose to use HepG2 cells to study the function of ARD1. ARD1 short hairpin RNA delivered by a retroviral vector caused >80% reduction in ARD1 message. We observed decreases in erythropoietin and vascular endothelial growth factor protein production, whereas there was no change in the HIF-1alpha protein level. A gene chip analysis of HepG2 cells transduced with virus expressing ARD1 short hairpin RNA under normoxia and hypoxia conditions or with virus overexpressing recombinant ARD1 confirmed that inhibition of ARD1 does not cause activation of HIF and downstream target genes. However, this analysis revealed that ARD1 is involved in cell proliferation and in regulating a series of cellular metabolic pathways that are regulated during hypoxia response. The role of ARD1 in cell proliferation is confirmed using fluorescence labeling analysis of cell division. From these studies we conclude that ARD1 is not required to suppress HIF but is required to maintain cell proliferation in mammalian cells.

Acetyltransferases↗
PubMed2002

RNA interference: the fragile X syndrome connection.

Fragile X syndrome is caused by loss of expression of FMRP, a protein proposed to act as a regulator of mRNA translation which promotes synaptic maturation and function. Now FMRP has been found to associate with the RNP complex that mediates post-transcriptional silencing by RNAi.

Animals↗
PubMed2004

RNA interference suggests a primary role for monoacylglycerol lipase in the degradation of the endocannabinoid 2-arachidonoylglycerol.

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is produced by neurons and other cells in a stimulus-dependent manner and undergoes rapid biological inactivation through transport into cells and catalytic hydrolysis. The enzymatic pathways responsible for 2-AG degradation are only partially understood. We have shown previously that overexpression of monoacylglycerol lipase (MGL), a cytosolic serine hydrolase that cleaves 1- and 2-monoacylglycerols to fatty acid and glycerol, reduces stimulus-dependent 2-AG accumulation in primary cultures of rat brain neurons. We report here that RNA interference-mediated silencing of MGL expression greatly enhances 2-AG accumulation in HeLa cells. After stimulation with the calcium ionophore ionomycin, 2-AG levels in MGL-silenced cells were comparable with those found in cells in which 2-AG degradation had been blocked using methyl arachidonyl fluorophosphonate, a nonselective inhibitor of 2-AG hydrolysis. The results indicate that MGL plays an important role in the degradation of endogenous 2-AG in intact HeLa cells. Furthermore, immunodepletion experiments show that MGL accounts for at least 50% of the total 2-AG-hydrolyzing activity in soluble fractions of rat brain, suggesting that this enzyme also contributes to 2-AG deactivation in the central nervous system.

Animals↗