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Results for “RNA interference”

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At least 1,117 records · Page 62Linked to original sources

PubMed2000

Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals.

Double-stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence-specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co-bombarded UidA (GUS) and TaGLP2a:GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol-4-reductase in maize and barley, respectively. Dihydroflavonol-4-reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1- and b-Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol-4-reductase gene, C1- and b-Peru-dependent, cell-autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non-specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss-of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence-specific interference with gene function at the single-cell level.

Alcohol Oxidoreductases↗

PubMed2005

CD2AP expression in a renal ischemia/reperfusion injury model and analysis of its related genes using overexpression and RNA interference technique.

BACKGROUND: CD2-associated protein (CD2AP) is a ubiquitously expressed 80-kDa intracellular protein, and has been speculated to act as an intracellular signaling pathway between plasma membrane proteins and cytoskeleton proteins. CD2AP expression has been reported in both the glomerulus and tubular epithelium in the kidney, and CD2AP knockout mice exhibit congenital nephrotic syndrome. However, the precise properties and its role in the renal tubules have not been clarified. METHODS: We used an established rat model of ischemic/reperfusion renal injury (IRI) to examine the expression of CD2AP by real-time PCR, Western blotting, and immunohistochemistry. We also investigated the expression of genes related to apoptosis and cell proliferation in mouse collecting duct-derived cells (M1 cells) transfected with full-length of CD2AP cDNA or short interfering RNA. RESULTS: CD2AP mRNA and protein expression had significantly increased in the IRI kidney. Real-time PCR indicated that expression of genes regulating apoptosis, such as B-Raf and Caspase-12, and genes regulating cell proliferation factors, CDC2, was decreased in CD2AP-overexpressing M1 cells and increased in CD2AP-interfered M1 cells. CONCLUSIONS: These results suggest that CD2AP expression was increased following renal ischemia and that CD2AP may be related to the process of cell repair and/or cell differentiation following injury.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Femcoat, a novel eggshell protein in Drosophila: functional analysis by double stranded RNA interference.

In Drosophila oogenesis, follicle cells derived from somatic tissue surround the oocyte and play key roles in generating properly polarized oocytes. During the later steps of oogenesis, follicle cells are involved in secretion of proteins that make the eggshell, an essential protective layer for the oocyte. Although studies on the signaling processes to make polarized oocytes have been progressed very far, studies on the mechanisms for eggshell formation is not clear yet. To elucidate the underlying mechanism in eggshell formation, we used a differential display screen to isolate genes that are specifically expressed during the later stages of oogenesis, and isolated a novel gene, Femcoat. Femcoat encodes a putative chorion membrane protein that contains many highly charged residues and has a putative signal peptide. Femcoat is expressed specifically in the follicle cells with a punctate staining pattern typical of secreted proteins, and becomes cross-linked heavily at the final steps of oogenesis. To identify the developmental role of Femcoat in eggshell formation, we performed an inducible double stranded RNA mediated interference (dsRNAi) method to specifically reduce Femcoat expression during oogenesis in adult flies. Electron microscopy analysis of egg chambers from these flies showed defects in chorion formation. These pieces of evidence demonstrated that Femcoat is necessary for eggshell formation, especially during chorion synthesis. Our results demonstrate that inducible dsRNAi analysis can be effective in determining the developmental function of novel genes.

Amino Acid Sequence↗

PubMed2007

flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference.

The potato cyst nematode Globodera pallida is a serious pest of potato crops. Nematode FMRFamide-like peptides (FLPs) are one of the most diverse neuropeptide families known, and modulate sensory and motor functions. As neuromuscular function is a well-established target for parasite control, parasitic nematode FLP signaling has significant potential in novel control strategies. In the absence of transgenic parasitic nematodes and the reported ineffectiveness of neuronal gene RNAi in Caenorhabditis elegans, nothing is known about flp function in nematode parasites. In attempts to evaluate flp function in G. pallida, we have discovered that, unlike in C. elegans, these genes are readily susceptible to RNAi. Silencing any of the five characterized G. pallida flp genes (Gp-flp-1, -6, -12, -14, or -18) incurred distinct aberrant behavioral phenotypes consistent with key roles in motor function. Further delineation of these effects revealed that double-stranded RNA exposure time (> or = 18 h) and concentration (> or = 0.1 microg/ml) were critical to the observed effects, which were reversible. G. pallida flp genes are essential to coordinated locomotory activities, do not display redundancy, and are susceptible to RNAi, paving the way for the investigation of RNAi-mediated flp gene silencing as a novel plant parasite control strategy.

Animals↗

PubMed2006

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression.

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

Animals↗

PubMed2006

RNA interference of Hemolin causes depletion of phenoloxidase activity in Hyalophora cecropia.

Melanization is regulated by the prophenoloxidase cascade and functions as a response to intruding microorganisms in invertebrates. When injecting dsRNA of the lepidopteran immune protein hemolin in pupae of Hyalophora cecropia (Lepidoptera: Saturniidae), we observed a significant reduction in phenoloxidase activity after 24 h, but not after 72 h. The link between hemolin and the prophenoloxidase system suggests that hemolin is a pattern recognition protein important for the triggering of the prophenoloxidase cascade in the defence against bacterial infections.

Animals↗

PubMed2004

RETRACTED: Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: A novel therapeutic strategy in pancreatic cancer.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editors-in-Chief, because an investigation by Harvard Medical School and the Brigham and Women’s Hospital has concluded that Figure 1A is invalid and no underlying research data are available to resolve the discrepancies or validate the reported results.

Apoptosis↗

PubMed2003

Bax inhibitor-1 is overexpressed in prostate cancer and its specific down-regulation by RNA interference leads to cell death in human prostate carcinoma cells.

To analyze differential gene expression of putative prostate tumor markers we compared the expression levels of more than 400 cancer-related genes using the cDNA array technique in a set of capsule-invasive prostate tumor and matched normal prostate tissue. The overexpression of Bax inhibitor-1 (BI-1) in prostate carcinoma and prostate cancer cell lines was confirmed by using Northern blot and Western blot analyses. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) on intact RNAs from 17 paired laser-captured microdissected epithelial tissue samples confirmed up-regulated BI-1 expression in 11 of 17 prostate tumors. In addition, it was demonstrated that BI-1 expression is down-regulated in stromal cells as compared to matched normal epithelial cells of the prostate. In situ hybridization experiments on prostate sections also revealed that BI-1 expression is mainly restricted to epithelial cells. Furthermore, quantitative RT-PCR on RNAs derived from five benign prostate hyperplasia (BPH) samples showed no significant difference in BI-1 expression as compared to normal epithelial prostate tissue. To determine the function of BI-1 in vitro, human PC-3, LNCaP, and DU-145 prostate carcinoma cells were transfected with small interfering double-strand RNA (siRNA) oligonucleotides against the BI-1 gene leading to a specific down-regulation of BI-1 expression. Furthermore, transfection of PC-3, LNCaP, and DU-145 cells with BI-1 sequence-specific siRNAs caused a significant increase in spontaneous apoptosis in all cell lines. Taken together, our results indicate that the human BI-1 gene contains the potential to serve as a prostate cancer expression marker and as a potential target for developing therapeutic strategies for prostate cancer.

Apoptosis Regulatory Proteins↗

PubMed2004

[Inhibition of hepatitis B virus replication by RNA interference in vitro].

OBJECTIVE: To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that targets HBV core gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro. METHODS: HepG2 2.2.15 was used as target cells. The plasmid and liposome metafectene were cotransfected into the cultured cells, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR. RESULTS: The plasmid expressing siRNA was successfully constructed. The two constructed siRNAs could effectively inhibit HBV replication, and their inhibitive effect on HBV was dose-dependent. CONCLUSION: These results showed that siRNA could substantially inhibit HBV replication in the infected cells

Hepatitis B virus↗

PubMed2004

RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age.

Sequencing of complete genomes has provided researchers with a wealth of information to study genome organization, genetic instability, and polymorphisms, as well as a knowledge of all potentially expressed genes. The identification of all genes encoded in the human genome opens the door for large-scale systematic gene silencing using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs). With the recent development of siRNA and shRNA expression libraries, the application of RNAi technology to assign function to cancer genes and to delineate molecular pathways in which these genes affect in normal and transformed cells, will contribute significantly to the knowledge necessary to develop new and also improve existing cancer therapy.

Animals↗

PubMed2005

Inverted repeat PCR for the rapid assembly of constructs to induce RNA interference.

Expressing stem-loop RNAs in plants, fungi, and animals efficiently silences homologous target gene expression. We devised a novel PCR strategy, called inverted repeat PCR (IR-PCR), which allows rapid assembly and cloning of stem-loop-containing constructs in any vector. IR-PCR relies on differentially tagging antisense and sense copies of the target in one round of PCR and assembling them in a second. We used IR-PCR to assemble constructs targeting profilin, actin, and actin-related protein (ARP) transcripts from Arabidopsis. Immunoblotting of lines expressing a profilin PRF1 3' untranslated region (UTR)-specific construct demonstrated a 77 to 97% reduction in PRF1 protein, but not other profilin isovariants.

Base Sequence↗

PubMed2004

RNA interference demonstrates a novel role for H2A.Z in chromosome segregation.

The histone variant H2A.Z plays an essential role in metazoans but its function remains to be determined. Here, we developed a new inducible RNAi strategy to elucidate the role of H2A.Z in mammalian cell lines. We show that in the absence of H2A.Z, the genome becomes highly unstable and that this instability is caused by defects in the chromosome segregation process. Analysis of H2A.Z localization reveals that in these cells it is enriched at heterochromatic foci with HP1alpha on the arms of chromosomes but not at centromeric regions. When H2A.Z is depleted, normal HP1alpha-chromatin interactions are disrupted on the chromosomal arms and, notably, also at pericentric regions. Therefore, H2A.Z controls the localization of HP1alpha. We conclude that H2A.Z is essential for the accurate transmission of chromosomes.

Base Sequence↗

PubMed2005

Intracellular antibodies (intrabodies) versus RNA interference for therapeutic applications.

Currently there are two technology platforms for the ablation of protein function: (a) RNAi-mediated gene-silencing at the post-transcriptional level, and (b) intrabody knockout of protein function at the post-translational level. Both approaches hold much promise for therapeutic applications. The pertinent question is how to choose between these alternative approaches. This commentary examines the advantages and disadvantages of these newly-emerging technology platforms. The RNAi approach is much less technically challenging than the intrabody-mediated knockout of protein function, but its major limitation is non-specificity. Although it is time-consuming and labor-intensive to generate intrabodies for specific intracellular protein targets, a much higher level of specificity can be attained. Ultimately, the choice between these strategies depend on the specific application in question, as well as on further technical advances in both technology platforms.

Antibodies↗

PubMed2005

Long-term in vivo and in vitro AAV-2-mediated RNA interference in rat retinal ganglion cells and cultured primary neurons.

Viral vector-based expression of small interfering RNAs is a promising tool for gene regulation, both in cultured cells and in animal models. In this study, we analysed the ability of adeno-associated virus-2 to function as an RNAi vector in cultured primary hippocampal neurons in vitro and in retinal ganglion cells in vivo. We demonstrate a long-lasting, highly efficient, and specific down-regulation of gene expression in vivo and in vitro by the use of bicistronic vectors. This is the first evidence of a cell type-specific long-term (more than three-month-long) RNAi in the eye. Furthermore, our results constitute the prerequisite for the use of this technique in models of neurodegeneration and neuroregeneration in vivo and in vitro.

Animals↗

PubMed2004

Transgenic RNA interference reveals role for mouse sperm phospholipase Czeta in triggering Ca2+ oscillations during fertilization.

A sperm-specific phospholipase (PL) C, termed PLCzeta, is proposed to be the soluble sperm factor that induces Ca(2+) oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCzeta mRNA have reduced amounts of PLCzeta protein. Sperm derived from these transgenic mice trigger patterns of Ca(2+) oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca(2+) oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCzeta is the physiological trigger of Ca(2+) oscillations required for activation of development.

Animals↗

PubMed2006

RNA interference targeting Akt promotes apoptosis in hypoxia-exposed human neuroblastoma cells.

Overactivation of the PI3 kinase/Akt pathway plays an essential role in the development and progression of various tumors. Akt is a key component of this pathway and hyperactivated in different tumors including neuroblastoma and glioma. In the present study, we tested the therapeutic efficacy of siRNA targeting Akt in inducing apoptotic cell death in NBFL cells (a human neuroblastoma cell line) subjected to anoxia/reoxygenation (A/R), a process that has been shown to modulate growth and progression of malignant tumors. We observed that siRNA targeting Akt effectively induced apoptotic cell death in NBFL cells (as determined by TUNEL assay and activated caspase-3 immunoreactivity) under normoxic conditions, an effect that was greatly enhanced under conditions of A/R. These findings underscore the importance of Akt signaling in promoting survival of neuroblastoma cells and may have potential therapeutic applications.

Apoptosis↗

PubMed2006

[Obtaining new germplast of Brassica napus with high oleic acid content by RNA interference and marker-free transformation of Fad2 gene].

A seed-specific Fad2 gene expression cassette, which is free-marker gene and with sense and antisense structure, was constructed by using the promoter and terminator of rape seed storage protein cruciferin gene. Transgenic rape plants without selection marker genes were obtained by Agrobacterium-mediated transformation. The oleic acid content of transgenic plant seeds is 83.9%, which is nearly the same as that of Brassica napus with double Fad2 mutation (85%). The result of RT-PCR analysis shows that the raising of oleic acid content may be due to the degradation of Fad2 mRNA induced by co-transformation of sense-antisense Fad2 gene. These transgenic plants with high oleic acid trait grew normally and without the disadvantageous agronomic traits such as weak cold resistance, tardy development, death of buds and low rate of seed setting caused by Fad2 inactivation in mutant Brassica napus plants. This work would serve as a good base for breeding of more lines with high oleic acid content.

Brassica napus↗

PubMed2002

Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential nuclear cofactor involved in Notch signal transduction.

We used the UAS/GAL4 two component system to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi we demonstrate that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi. Our results demonstrate that inducible RNAi is a powerful tool to study the role of essential genes throughout development.

ATP-Binding Cassette Transporters↗