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At least 325 records · Page 18Linked to original sources
PubMed2006

Identification of Heterodera glycines (soybean cyst nematode [SCN]) cDNA sequences with high identity to those of Caenorhabditis elegans having lethal mutant or RNAi phenotypes.

The soybean cyst nematode (SCN; Heterodera glycines) is a devastating obligate parasite of Glycine max (soybean) causing one billion dollars in losses to the US economy per year and over ten billion dollars in losses worldwide. While much is understood about the pathology of H. glycines, its genome sequence is not well characterized or fully sequenced. We sought to create bioinformatic tools to mine the H. glycines nucleotide database. One way is to use a comparative genomics approach by anchoring our analysis with an organism, like the free-living nematode Caenorhabditis elegans. Unlike H. glycines, the C. elegans genome is fully sequenced and is well characterized with a number of lethal genes identified through experimental methods. We compared an EST database of H. glycines with the C. elegans genome. Our goal was identifying genes that may be essential for H. glycines survival and would serve as an automated pipeline for RNAi studies to both study and control H. glycines. Our analysis yielded a total of nearly 8334 conserved genes between H. glycines and C. elegans. Of these, 1508 have lethal phenotypes/phenocopies in C. elegans. RNAi of a conserved ribosomal gene from H. glycines (Hg-rps-23) yielded dead and dying worms as shown by positive Sytox fluorescence. Endogenous Hg-rps-23 exhibited typical RNA silencing as shown by RT-PCR. However, an unrelated gene Hg-unc-87 did not exhibit RNA silencing in the Hg-rps-23 dsRNA-treated worms, demonstrating the specificity of the silencing.

Animals↗
PubMed2005

Genome-wide application of RNAi to the discovery of potential drug targets.

Progress is being made in the development of RNA interference-based (RNAi-based) strategies for the control of gene expression. It has been demonstrated that small interfering RNAs (siRNAs) can silence the expression of target genes in a sequence-specific manner in mammalian cells. Various groups, including our own, have developed systems for vector-mediated specific RNAi. Vector-based siRNA- (or shRNA) expression libraries directed against the entire human genome and siRNA libraries based on chemically synthesized oligonucleotides now allow the rapid identification of functional genes and potential drug targets. Use of such libraries will enhance our understanding of numerous biological phenomena and contribute to the rational design of drugs against heritable, infectious and malignant diseases.

Animals↗
PubMed2006

Gene silencing in mosquito salivary glands by RNAi.

Salivary glands are the ultimate site of development in the insect of mosquito born pathogens such as Plasmodium. Mosquito salivary glands also secrete components involved in anti-haemostatic activities and allergic reactions. We investigated the feasibility of RNAi as a tool for functional analysis of genes expressed in Anopheles gambiae salivary glands. We show that specific gene silencing in salivary glands requires the use of large amounts of dsRNA, condition that differs from those for efficient RNAi in other mosquito tissues. Using this protocol, we demonstrated the role of AgApy, which encodes an apyrase, in the probing behaviour of An. gambiae.

Animals↗
PubMed2004

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Adenovirus E1A Proteins↗
PubMed2006

RNAi studies reveal a conserved role for RXR in molting in the cockroach Blattella germanica.

Ecdysteroids play a major role during developmental growth in insects. The more active form of these hormones, 20-hydroxyecdysone (20E), acts upon binding to its heterodimeric receptor, formed by the two nuclear receptors, EcR and RXR/USP. Functional characterization of USP has been exclusively conducted on the holometabolous insect Drosophila melanogaster. However, it has been impossible to extend such analysis to primitive-hemimetabolous insects since species of this group are not amenable to genetic analysis. The development of methodologies based on gene silencing using RNA interference (RNAi) after treatment with double-stranded RNA (dsRNA) in vivo has resolved such limitations. In this paper, we show that injection of dsRNA into the haemocoel of nymphs and adults of the cockroach Blattella germanica can be used to silence gene function in vivo. In our initial attempt to test RNAi techniques, we halted the expression of the adult-specific vitellogenin gene. We then used the same technique to silence the expression of the B. germanica RXR/USP (BgRXR) gene in vivo during the last nymphal instar. BgRXR knockdown nymphs progressed through the instar correctly but they arrested development at the end of the stage and were unable to molt into adults. The results described herein suggest that RXR/USP function, in relation to molting, is conserved across the insect Class.

Animals↗
PubMed2004

Survival of motor neuron gene downregulation by RNAi: towards a cell culture model of spinal muscular atrophy.

Gene silencing with double-stranded RNA (RNAi) has proved useful for gene function studies, and should be especially well suited to studying diseases resulting in embryonal lethality where transgenic animal models are difficult to generate. We are applying this approach to the autosomal recessive disease spinal muscular atrophy (SMA). SMA is caused by mutations in the survival of motor neuron gene (SMN). The SMN protein is ubiquitously expressed and plays a role in RNA processing and its reduction in SMA ultimately leads to motor neuron degeneration in the spinal cord. The reasons for this motor neuron selectivity, however, are still unclear. SMN is essential for the viability of most eukaryotic organisms and this has made the generation of animal models of SMA extremely difficult. Here we describe a different approach to study SMN function using RNAi to silence SMN expression in cells. We designed double-stranded small interfering RNA (siRNA) targeted against murine Smn and transfected the murine embryonal terato-carcinoma cell line P19. The siRNAs reduced both Smn RNA and protein levels in the P19 cells compared to controls. These results illustrate that double-stranded RNA can be an effective gene silencing approach even in a protein that is essential for survival and highly expressed, and it could therefore be a valuable tool to study SMN function.

Animals↗
PubMed2005

RNAi-mediated silencing of leptin gene expression increases cell death in C6 glioblastoma cells.

We previously demonstrated that the brain, pituitary, and C6 glioblastoma cells express leptin. To determine the physiological role of brain-derived leptin, we sought to selectively silence its expression using RNA interference (RNAi) in vitro. One of four potential targets, siRNA L7, reduced leptin mRNA by 50% (P < 0.05) and protein by 55% (P < 0.0001) in C6 cells. RNAi also induced a twofold increase in cell death as seen by ethidium homodimer-1 (P < 0.015) and TUNEL (P < 0.005) staining. These data suggest that endogenous leptin may be a critical factor promoting cell survival in the brain.

Analysis of Variance↗
PubMed2006

RNAi screen in mouse astrocytes identifies phosphatases that regulate NF-kappaB signaling.

Regulation of NF-kappaB activation is controlled by a series of kinases; however, the roles of phosphatases in regulating this pathway are poorly understood. We report a systematic RNAi screen of phosphatases that modulate NF-kappaB activity. Nineteen of 250 phosphatase genes were identified as regulators of NF-kappaB signaling in astrocytes. RNAi selectively regulates endogenous chemokine and cytokine expression. Coimmunoprecipitation identified associations of distinct protein phosphatase 2A core or holoenzymes with the IKK, NF-kappaB, and TRAF2 complexes. Dephosphorylation of these complexes leads to modulation of NF-kappaB transcriptional activity. In contrast to IKK and NF-kappaB, TRAF2 phosphorylation has not been well elucidated. We show that the Thr117 residue in TRAF2 is phosphorylated following TNFalpha stimulation. This phosphorylation process is modulated by PP2A and is required for TRAF2 functional activity. These results provide direct evidence for TNF-induced TRAF2 phosphorylation and demonstrate that phosphorylation is regulated at multiple levels in the NF-kappaB pathway.

Animals↗
PubMed2006

Allele-specific RNAi selectively silences mutant SOD1 and achieves significant therapeutic benefit in vivo.

RNA interference (RNAi) has the potential to treat diseases caused by dominant, gain-of-function type of gene mutations. In these diseases, one allele is mutated and produces a toxic protein, whereas the other allele is normal and performs vital functions. One challenge in the treatment is to specifically inhibit the mutant allele toxicity while maintaining the normal allele function. To test allele-specific silencing in vivo, we made transgenic mice that express an shRNA against mutant Cu, Zn superoxide dismutase gene (SOD1(G93A)), which causes amyotrophic lateral sclerosis (ALS) by a gain of an unknown toxic property. By crossing this transgenic line with mice that express SOD1(G93A) and mice that express wild-type human SOD1, we found that this shRNA specifically silences the mutant, but not the wild-type SOD1. The silencing of the mutant significantly delayed ALS onset and extended survival. Thus, RNAi can achieve allele-specific silencing and therapeutic benefit in vivo.

Alleles↗
PubMed2006

Knockdown of spalt function by RNAi causes de-repression of Hox genes and homeotic transformations in the crustacean Artemia franciscana.

Hox genes play a central role in the specification of distinct segmental identities in the body of arthropods. The specificity of Hox genes depends on their restricted expression domains, their interaction with specific cofactors and selectivity for particular target genes. spalt genes are associated with the function of Hox genes in diverse species, but the nature of this association varies: in some cases, spalt collaborates with Hox genes to specify segmental identities, in others, it regulates Hox gene expression or acts as their target. Here we study the role of spalt in the branchiopod crustacean Artemia franciscana. We find that Artemia spalt is expressed in the pre-segmental 'growth zone' and in stripes in each of the trunk (thoracic, genital and post-genital) segments that emerge from this zone. Using RNA interference (RNAi), we show that knocking down the expression of spalt has pleiotropic effects, which include thoracic to genital (T-->G), genital to thoracic (G-->T) and post-genital to thoracic (PG-->T) homeotic transformations. These transformations are associated with a stochastic de-repression of Hox genes in the corresponding segments of RNAi-treated animals (AbdB for T-->G and Ubx/AbdA for G-->T and PG-->T transformations). We discuss a possible role of spalt in the maintenance of Hox gene repression in Artemia and in other animals.

Animals↗
PubMed2006

A new inducible RNAi xenograft model for assessing the staged tumor response to mTOR silencing.

Human xenograft tumor models are widely used for efficacy evaluation of potential cancer targets. siRNA is usually stably introduced into tumor cells prior to transplantation. However, silencing of the cancer therapeutic target usually results in reduced cell growth/survival in vitro and/or failure to establish tumors in vivo, thus hindering tumor response-based efficacy evaluation. The present study explored a new tumor response model based on regulated RNAi, which is more relevant from a clinical standpoint. As a proof of principle, an inducible lentiviral RNAi vector was used to silence the known cancer therapeutic target mTOR upon induction with Doxycycline (DOX). The responses to DOX-induced mTOR silencing were tested both in vitro and in vivo for prostate cancer PC3 models. Significant reduction in cancer cell survival was observed due to cell cycle arrest and apoptosis when mTOR silencing was induced in vitro. mTOR silencing also caused tumor regression for the early-staged PC3 tumors (100% tumor regressed and 45% became tumor-free). The advanced-staged tumors also demonstrated significant responses (100% regressed). Therefore, our results demonstrate the powerful utility of this new inducible xenograft tumor model for efficacy evaluation of cancer targets, and it provides a direct in vivo efficacy validation of mTOR as a cancer therapeutic target.

Animals↗
PubMed2005

Amplification of RNAi--targeting HLA mRNAs.

Posttranscriptional suppression of gene expression can be achieved by introduction of sequence-specific small interfering (si) RNA duplexes and by de novo intracellular synthesis of short sequence-specific double-stranded RNAs. However, achieving desired levels of knockdown is a barrier to successful analytic and therapeutic application. We demonstrate that increasing expression of introduced short hairpin RNA (shRNA) can markedly enhance RNA interference (RNAi) and that this approach can be used to achieve maximal target down-regulation, when the choice of optimal siRNA-binding sites is restricted or when multiple genes are simultaneously targeted and the amount of siRNA is limiting. A dose-dependent RNAi effect was accomplished by placing copies of shRNA under control of the Pol III U6 small nuclear RNA promoter in tandem in a DNA vector. Using this system, we achieved simultaneous down-regulation of expression of classical human leukocyte antigen (HLA) class I genes in cultured and primary human T cells, which might be applied to help circumvent T-cell-mediated rejection of immunogenic and/or HLA-disparate allografts.

Base Sequence↗
PubMed2003

Efficient and specific down-regulation of prion protein expression by RNAi.

Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challenge by the infectious agent. These results indicated that a down-regulation of the Prnp gene expression is a potential therapeutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specifically reduce the level of PrPc in transfected cells. It, thus, indicates that RNAi is an attractive therapeutic approach to fight against prion diseases.

Animals↗
PubMed2003

Transgenic RNAi in mouse oocytes: a simple and fast approach to study gene function.

Double-strand RNA (dsRNA)-mediated posttranscriptional gene silencing, also known as RNA interference (RNAi), is a powerful tool to inhibit gene expression in several experimental model systems, including Arabidopsis, Caenorhabditis, and Drosophila. We previously described that the microinjection of Mos dsRNA into fully grown mouse oocytes results in the specific degradation of Mos mRNA in a time- and concentration-dependent manner. We report here a transgenic RNAi approach that is suitable to study gene function during mouse oocyte development and differentiation. The oocyte-specific Zp3 promoter was used to drive the expression of a long hairpin dsRNA ( approximately 500 bp) targeting Mos mRNA. Transgenic founder animals appeared healthy, but while males were fertile, females were not, in accordance with the known Mos null phenotype. The amount of Mos mRNA in the transgenic F(1) females was reduced by >90%, whereas there was no decrease in the nontargeted tissue plasminogen activator (Plat) mRNA. Moreover, the maturation-associated increase in mitogen-activated protein (MAP) kinase activity was not observed, and the metaphase II eggs underwent spontaneous parthenogenetic activation, thus recapitulating the Mos null phenotype. This approach provides a powerful method to study the functions of any oocyte-synthesized gene during oocyte development and early embryogenesis.

Animals↗
PubMed2006

Harnessing RNAi for analyses of Ras signaling and transformation.

The Ras-regulatory network is a loosely defined composition of numerous Ras family members and effector pathways that couple to critical cell-regulatory processes. Investigators are increasingly turning to RNAi-mediated inhibition of gene expression as an effective tool to help generate authentic portraits of Ras protein function in general and to accurately characterize the contribution of Ras family members and Ras effectors to oncogenic transformation in particular. Here we provide detailed protocols for high-efficiency and high-throughput delivery of siRNAs to human cancer cell lines and primary human epithelial cells. In addition, we discuss appropriate controls and limitations for the use of RNAi to derive biologically relevant observations.

Cell Line, Tumor↗
PubMed2001

RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs.

In posttranscriptional gene silencing (PTGS), "quelling," and RNA interference (RNAi), 21-25 nucleotide RNA fragments are produced from the initiating dsRNA. These short interfering RNAs (siRNAs) mediate RNAi by an unknown mechanism. Here, we show that GFP and Pp-Luc siRNAs, isolated from a protein complex in Drosophila embryo extract, target mRNA degradation in vitro. Most importantly, these siRNAs, as well as a synthetic 21-nucleotide duplex GFP siRNA, serve as primers to transform the target mRNA into dsRNA. The nascent dsRNA is degraded to eliminate the incorporated target mRNA while generating new siRNAs in a cycle of dsRNA synthesis and degradation. Evidence is presented that mRNA-dependent siRNA incorporation to form dsRNA is carried out by an RNA-dependent RNA polymerase activity (RdRP).

Animals↗
PubMed2001

RNAi and brain function: was McConnell on the right track?

RNA interference (RNAi), one of the hottest topics of molecular biology research today, has unique features that are eerily reminiscent of the phenomenon of "RNA-mediated memory transfer," a controversial line of work that was investigated with great enthusiasm in the 1960s. If not a coincidence, then this suggests taking a new look at RNA-mediated modulation of neural function and raises the possibility that RNAi might be one of the physiologic mechanisms that regulate long-term gene expression in the brain.

Animals↗
PubMed2002

RNA interference (RNAi) inhibits growth of Plasmodium falciparum.

RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. Erythrocyte-infecting stages of the malaria parasite Plasmodium falciparum were treated with double-stranded RNA (dsRNA) encoding a segment of the gene encoding dihydroorotate dehydrogenase (DHODH). DHODH is an enzyme in pyrimidine biosynthesis, essential for parasite growth. A decrease in parasite growth (P<0.0005) correlated with a decrease in levels of DHODH mRNA. Control treatments with single-stranded RNA, dsRNA encoding the circumsporozoite protein (a stage-specific protein not expressed in the asexual blood stage) and dsRNA encoding a gene from the related organism Toxoplasma gondii did not inhibit growth. As a test for the RNAi assay, parasites were treated with dsRNA encoding chorismate synthase (CS), an enzyme thought to be involved in folate synthesis, to examine the requirement for this enzyme for parasite growth. Growth decreased (P<0.001) though less markedly than by dsRNA encoding DHODH. These results demonstrate the utility of this assay in assessing requirements for gene products, and their potential as chemotherapeutic targets.

Animals↗