dsRNA

Distribution by Scientific Domains
Distribution within Life Sciences


Selected Abstracts


Endoplasmic reticulum stress and the unfolded protein response are linked to synergistic IFN-, induction via X-box binding protein 1

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2008
Judith A. Smith Dr.
Abstract Type,I IFN are strongly induced upon engagement of certain pattern recognition receptors by microbial products, and play key roles in regulating innate and adaptive immunity. It has become apparent that the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), in addition to restoring ER homeostasis, also influences the expression of certain inflammatory cytokines. However, the extent to which UPR signaling regulates type,I IFN remains unclear. Here we show that cells undergoing a UPR respond to TLR4 and TLR3 ligands, and intracellular dsRNA, with log-fold greater IFN-, induction. This synergy is not dependent on autocrine type,I IFN signaling, but unexpectedly requires the UPR transcription factor X-box binding protein,1 (XBP-1). Synergistic IFN-, induction also occurs in HLA-B27/human ,2m-transgenic rat macrophages exhibiting a UPR as a consequence of HLA-B27 up-regulation, where it correlates with activation of XBP-1 splicing. Together these findings indicate that the cellular response to endogenous ,danger' that disrupts ER homeostasis is coupled to IFN-, induction by XBP-1, which has implications for the immune response and the pathogenesis of diseases involving the UPR. [source]


Inhibitory effect of the polyinosinic-polycytidylic acid/cationic liposome on the progression of murine B16F10 melanoma

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2006
Taku Fujimura
Abstract Cellular proteins, retinoic acid inducible gene-I and Toll-like receptor 3, sense dsRNA including polyinosinic-polycytidylic acid (PIC) to stimulate innate immune response. The local administration of PIC has been demonstrated to be effective in anti-tumor immunotherapy. However, the effects of PIC delivered cross the cell membrane have not yet been examined. To address this issue, we used a complex of PIC and cationic liposome (PIC liposome) and examined its anti-tumor effects in vitro and in vivo. PIC liposome could directly suppress the growth of B16F10 melanoma in vitro and repeated peritumoral injections of PIC liposome inhibited melanoma growth in a dose-dependent manner. This treatment induced tyrosinase-related protein-2 (TRP-2)-tetramer+ CD8+ cells in the lymph nodes. As the mechanism for its anti-tumor immune response, we showed that the intradermal injection of PIC liposome induced the maturation of dendritic cells (DC). Moreover, the intratumoral injection of immature DC after treatment with PIC liposome significantly increased the number of TRP-2-specific IFN-,-producing cells in the lymph nodes as well as spleen, which resulted in an augmentation of the anti-tumor immune response. These studies demonstrate the potential of peritumoral injection of PIC liposome as immunotherapy for malignant melanoma. [source]


Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-, and provide a mechanism for enhanced synovial chemokine levels in septic arthritis

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2003
Paul Proost
Abstract The CXC chemokine IFN-,-inducible protein-10 (IP-10/CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T cells and natural killer cells. Peripheral blood mononuclearcells (PBMC) produce low but significant amounts of IP-10/CXCL10 protein upon stimulation with double-stranded (ds) RNA, the Toll-like receptor 3 (TLR3) ligand. IFN-, is a superior IP-10/CXCL10inducer. The bacterial TLR4 and TLR2 ligands, LPS and peptidoglycan (PGN), inhibit IFN-,- or dsRNA-dependent IP-10/CXCL10 production in PBMC, whereas IL-8/CXCL8 production was enhanced. In fibroblasts a different picture emerges with IFN-, inducing moderate and dsRNA provoking strong IP-10/CXCL10 production. Furthermore, treatment of fibroblasts with IFN-, in combination with bacterial LPS or PGN results in a synergistic production of IP-10/CXCL10 and IL-8/CXCL8. The synergistic induction of IP-10/CXCL10 in fibroblasts is reflected by significantly enhanced IP-10/CXCL10 concentrations in synovial fluids of septic compared to osteoarthritis patients to reach on average higher levels than those of IL-8/CXCL8. These high amounts of IP-10/CXCL10 produced by connective tissue fibroblasts not only attract CXCR3 expressing activated Th1 cells and natural killer cells to sites of infection but may also antagonize the CCR3 dependent attraction of Th2 lymphocytes and exert CXCR3-independent, defensin-like antibacterial activity. [source]


Targeted disruption of a pupal hemocyte protein of Sarcophaga by RNA interference

FEBS JOURNAL, Issue 20 2001
Takeshi Nishikawa
Previously, we purified a transmembrane protein with a molecular mass of 120 kDa (p120) that is exclusively expressed in pupal hemocytes of Sarcophaga. In this study, we demonstrated that double-stranded RNA (dsRNA) injected into the larval body cavity effectively inhibited the expression of p120 in pupal hemocytes. Thus, RNA interference (RNAi) was found to be a useful technique for creating pupal hemocytes with a loss-of-function of a specific protein. The p120-less pupal hemocytes generated by RNAi were found to have lost the ability to take up acetylated low density lipoprotein, indicating that p120 is a scavenger receptor specifically expressed on the surface of pupal hemocytes. [source]


Interferon-, synergistically enhances induction of interleukin-6 by double stranded RNA in HeLa cells

FEBS JOURNAL, Issue 9 2000
Jennifer L. Harcourt
Double stranded RNA (dsRNA), an intermediate that is common during viral infection, directly induces much higher levels of expression of interleukin-6 (IL-6) mRNA than does the cytokine IL-1,. Interferon , (IFN,) by itself does not induce expression of IL-6; nonetheless, IFN, pretreatment dramatically enhances IL-6 induction by dsRNA but not by IL-1,. Mutation of either the activating transcription factor/cyclic AMP response element binding protein (ATF/CREB) or the NF-IL-6 binding element within the IL-6 promoter eliminates most responsiveness of CAT reporter constructs to either dsRNA or to IL-1,. IFN, pretreatment partially restores responsiveness to dsRNA but not to IL-1, when either the ATF/CREB site or the NF-IL-6 site is mutated, but at least one of these sites must be intact for responsiveness to be restored. Mutation of the ,B binding site in the IL-6 promoter eliminates responsiveness to either IL-1, or to dsRNA, and pretreatment with IFN, does not restore any responsiveness. Incubation with dsRNA leads to a decrease in protein translation, especially in cells that have been pretreated with IFN,. Nonetheless, IFN, pretreatment followed by dsRNA leads to very high IL-6 protein levels. These studies demonstrate that major differences exist in the induction of IL-6 at both the mRNA and protein levels by dsRNA compared to cytokines and that IFN, pretreatment selectively enhances IL-6 induction by dsRNA but not by IL-1,. The high levels of IL-6 expression that result when cells encounter class I IFN prior to dsRNA suggest a mechanism for a heightened host response to viral infection with heightened production of this pleotropic cytokine. [source]


Rhinovirus increases human ,-defensin-2 and -3 mRNA expression in cultured bronchial epithelial cells

FEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2003
Louise A Duits
Abstract Human ,-defensins (hBDs) are antimicrobial peptides that play important roles in host defense against infection, inflammation and immunity. Previous studies showed that micro-organisms and proinflammatory mediators regulate the expression of these peptides in airway epithelial cells. The aim of the present study was to investigate the modulation of expression of hBDs in cultured primary bronchial epithelial cells (PBEC) by rhinovirus-16 (RV16), a respiratory virus responsible for the common cold and associated with asthma exacerbations. RV16 was found to induce expression of hBD-2 and -3 mRNA in PBEC, but did not affect hBD-1 mRNA. Viral replication appeared essential for rhinovirus-induced ,-defensin mRNA expression, since UV-inactivated rhinovirus did not increase expression of hBD-2 and hBD-3 mRNA. Exposure to synthetic double-stranded RNA (dsRNA) molecule polyinosinic:polycytidylic acid had a similar effect as RV16 on mRNA expression of these peptides in PBEC. In line with this, PBEC were found to express TLR3, a Toll-like receptor involved in recognition of dsRNA. This study shows that rhinovirus infection of PBEC leads to increased hBD-2 and hBD-3 mRNA expression, which may play a role in both the uncomplicated common cold and in virus-associated exacerbations of asthma. [source]


Incomplete movement of Cryphonectria hypovirus 1 within a vegetative compatibility type of Cryphonectria parasitica in natural cankers on grafted American chestnut trees

FOREST PATHOLOGY, Issue 6 2002
E. P. Hogan
Summary American chestnut trees, grafted in 1980 from large survivors, were inoculated in 1982 and 1983 with four white (European) hypovirulent strains of Cryphonectria parasitica, infected with C. hypovirus 1 (CHV1); this hypovirus has been shown to be capable of moving rapidly within the mycelium of a vegetative compatibility (vc) type of C. parasitica in blight cankers. Using a 49-cell lattice plot, 17.8×17.8 cm, the spatial patterns and frequencies of white and pigmented isolates and white and pigmented vc types were investigated within superficial cankers on the grafts located outside the hypovirulent-strain-inoculated zone. Four of six cankers assayed contained white isolates, and three of the four had random spatial patterns of white isolates, based on join-count statistics. Vc tests, using pigmented isolates and pigmented single-spore colonies of white isolates, indicated that the majority of white and pigmented isolates recovered from each of two cankers assayed were in one vc type. White and pigmented lattice-plot cells of the same vc type were frequently in contact with each other, indicating incomplete movement of CHV1 within a vc type. Nine and 10 vc types were found in the two cankers; it is hypothesized that small, white vc type areas in each canker may be a source of CHV1 transmission to the major vc types. Based on join-count statistics, the spatial pattern of the single, major vc type in one canker was non-random (aggregated), whereas the other canker had a random major vc type pattern. White and pigmented in vitro variants (sectors) of C. parasitica, that resemble white and pigmented in vivo variants in spatial contact and vc compatibility, were intermediate hypovirulent and virulent on forest American chestnuts, and dsRNA positive and negative, respectively. Incomplete movement of CHV1 within a vc type could be a major cause of the prevalence of pigmented isolates in superficial cankers on chestnut trees. Résumé Des châtaigniers américains greffés en 1980 à partir de grands arbres survivants, ont été inoculés en 1982 et 1983 avec quatre souches blanches (européennes) hypovirulentes de Cryphonectria parasitica, infectées par l'hypovirus 1 (CHV 1). Cet hypovirus avait été montré capable de migrer rapidement dans le mycélium végétativement compatible (vc) de C. parasitica dans des chancres. Grâce un dispositif à 49 cellules (17,8×17,8 cm), la répartition spatiale et la fréquence des isolats blancs et pigmentés, et des GCV, ont étéétudiées dans des chancres superficiels chez les greffons, en dehors de la zone inoculée par les souches hypovirulentes. Quatre des six chancres étudiés contenaient des isolats blancs, dont la répartition spatiale était erratique chez trois d'entre eux. Les tests de compatibilité végétative, utilisant des isolats pigmentés et des colonies pigmentées mono-sporées d'isolats blancs, ont montré que la majorité des isolats blancs et des isolats colorés trouvés dans chacun des deux chancres analysés, était d'un seul type végétatif. Les cellules du dispositif, blanches ou pigmentées du même GCV, étaient fréquemment en contact, ce qui indique un mouvement incomplet de CHV1 dans un GCV. Neuf et dix GCV ont été trouvés dans les deux chancres et on avance l'hypothèse que de petites zones à GCV blancs dans chaque chancre peuvent être une source de transmission de CHV1 aux types végétatifs majoritaires. L'unique GCV majoritaire dans un des chancres n'était pas réparti au hasard (agrégé), mais il l'était dans l'autre chancre. Les variants blancs et pigmentés in vitro (secteurs) de C. parasitica, qui ressemblent aux variants blancs et pigmentés in vivo en contact spatial, étaient intermédiaires en hypovirulence et en virulence sur les châtaigniers américains, et respectivement positifs et négatifs pour le dsRNA. Un mouvement incomplet de CHV1 dans un GCV pourrait être la principale cause de la prévalence d'isolats pigmentés dans des chancres superficiels. Zusammenfassung Amerikanische Kastanien (Castanea dentata), die im Jahr 1980 von adulten überlebenden Exemplaren veredelt worden waren, wurden 1982 und 1983 mit vier unpigmentierten, hypovirulenten Stämmen von Cryphonectria parasitica aus Europa inokuliert, die mit dem Cryphonectria parasitica - hypovirus 1 (CHV 1) infiziert waren. Es war nachgewiesen, dass sich dieses Hypovirus rasch innerhalb des Myzels eines vc-Typs von C. parasitica in Krebsen ausbreiten konnte. Bei oberflächlichen Krebsen an veredelten Bäumen ausserhalb der mit dem hypovirulenten Stamm inokulierten Zone wurde auf einer 17,8×17,8 cm grossen Fläche (die in 49 Quadrate unterteilt wurde) die räumliche Verteilung und die Häufigkeit von unpigmentierten und pigmentierten Isolaten sowie der vc-Typen untersucht. Vier von sechs analysierten Krebsen enthielten weisse Isolate und drei davon zeigten zufällige Verteilungsmuster der Isolate (gemäss Joint-Count-Statistik). Vc-Tests mit pigmentierten Isolaten und pigmentierten Einzelsporkulturen von weissen Isolaten zeigten, dass die Mehrheit der weissen und der pigmentierten Isolate von zwei Krebsen zur gleichen vc-Gruppe gehörten. Weisse und pigmentierte Probepunkte mit dem gleichen vc-Typ waren häufig benachbart, was eine unvollständige Ausbreitung des CHV 1 innerhalb eines vc-Typs anzeigt. In den 2 Krebsen wurden 9 bzw. 10 vc-Typen nachgewiesen und es wird vermutet, dass kleine Bereiche mit weissem vc-Typ innerhalb eines Krebses als Reservoir für die Übertragung des Virus zu den dominanten vc-Typen fungieren können. Mit Hilfe der Joint-Count-Statistik wurde nachgewiesen, dass das räumliche Muster des einen, dominanten vc-Typs in einem Krebs nicht zufällig, sondern aggregiert war, während der andere Krebs ein zufälliges Verteilungsmuster der vc-Typen aufwies. Weisse und pigmentierte Varianten von C. parasitica, die in vitro auftraten (Sektoren), und die den weissen und pigmentierten Varianten sehr ähnlich sind, die in der Natur in räumlichem Kontakt zueinander auftreten, und die vegetativ kompatibel sind, zeigten in Feldversuchen auf C. sativa eine mittlere Hypovirulenz bzw. Virulenz. Ausserdem waren sie dsRNA positiv bzw. negativ. Die unvollständige Ausbreitung des CHV 1 innerhalb einer vc-Typs könnte das überwiegen pigmentierter Isolate in oberflächlichen Krebsen erklären. [source]


Persistence of Cryphonectria hypoviruses after their release for biological control of chestnut blight in West Virginia forests

FOREST PATHOLOGY, Issue 6 2002
Y.-C. Liu
Summary Although Cryphonectria hypoviruses have been relatively successful as biological control agents of chestnut blight in Europe, their success in North America has been limited. Experimental releases of hypoviruses were made in 1978,82 at two sites in West Virginia forests with high densities of regenerating chestnut trees. Cryphonectria hypovirus 1 (CHV-1) from Europe, as well as American isolates of Cryphonectria parasitica containing CHV-3, were used for these releases. Although most trees died during the 5-year release period, it was not known if the hypoviruses persisted in the C. parasitica population at the two sites. When the experimental plots were revisited in 1994, few chestnut trees were found. The exception was one plot containing coppice sprouts that had grown from the root collars of the original trees. The authors intensively sampled C. parasitica from experimental plots and screened recovered isolates for double-stranded RNA (dsRNA). None of the isolates contained CHV-1; only six isolates contained CHV-3, all from the plots with the coppice sprouts. CHV-4, which occurs naturally in West Virginia forests and in two released isolates, hybridized to dsRNA from the isolates containing CHV-3, indicating mixed infections. CHV-4 also hybridized to dsRNA from other isolates sampled inside and outside the treated plots. In contrast to CHV-1 and CHV-3, however, CHV-4 has little effect on the growth or phenotype of C. parasitica. The limited persistence of CHV-1 and CHV-3 may have resulted when the C. parasitica population was reduced in size due to the failure of chestnut trees to resprout because of competition from other hardwood species. Résumé Bien que les hypovirus de Cryphonectria aient remporté un réel succès comme agents de lutte biologique contre le chancre du châtaignier en Europe, leur succès en Amérique du nord a été limité. Des applications d'hypovirus ont été faites en 1978,1982 en Virginie de l'ouest dans deux sites forestiers à fortes densités de régénérations de châtaigniers. Le CHV-1 originaire d'Europe ainsi que des isolats américains contenant le CHV-3 ont été utilisés dans ces essais. Bien que la plupart des arbres soient morts pendant les cinq ans de l'application, on ne sait pas si les hypovirus ont persisté dans la population de Cryphonectria parasitica. Quand les parcelles expérimentales ont été revisitées en 1994, peu de châtaigniers ont été trouvés. Un site faisait exception qui était un taillis de rejets issu de racines de souches. Nous avons échantillonné intensivement le C. parasitica dans les placettes et le dsRNA a été examiné dans les isolats obtenus. Parmi tous les isolats provenant des placettes de taillis, aucun ne contenait CHV-1 et seulement six contenaient CHV-3. CHV-4 qui existe naturellement dans les forêts en Virginie de l'ouest et dans deux des isolats appliqués, s'hybridait avec le dsRNA d'autres isolats contenant CHV-3, ce qui indique des infections mixtes. CHV-4 s'hybridait aussi avec le dsRNA d'autres isolats recueillis dans ou au dehors des placettes traitées. Contrairement à CHV1 et à CHV-3, CHV-4 a cependant peu d'effet sur la croissance et le phénotype de C. parasitica. La persistance limitée de CHV-1 et de CHV-3 peut avoir résulté de la réduction de la population de C. parasitica par un manque de rejets de châtaigniers dûà la compétition avec les autres espèces de feuillus. Zusammenfassung Währenddem der Einsatz von Cryphonectria Hypoviren zur biologischen Bekämpfung des Kastanienrindenkrebses in Europa relativ erfolgreich war, stimmt dies in Nordamerika nur begrenzt. In West-Virginia wurden in den Jahren 1978,82 Hypoviren in Experimenten an zwei Waldstandorten mit hoher Dichte regenerierender Edelkastanien freigesetzt. Cryphonectria hypovirus 1 (CHV-1) von Europa und amerikanische Isolate von Cryphonectria parasitica, die CHV-3 enthielten, wurden für diese Freisetzungsversuche verwendet. Obschon die meisten Bäume während der fünfjährigen Freisetzungsperiode abstarben, war unklar, ob die Hypoviren in der C. parasitica Population überlebten. Als die Versuchsflächen im Jahre 1994 erneut aufgesucht wurden, konnten nur noch wenige Edelkastanien gefunden werden mit Ausnahme einer Fläche, auf der die Wurzelanläufe ursprünglicher Bäume Stockausschläge trugen. Zahlreiche C. parasitica Stämme wurden von den Versuchsflächen gewonnen und auf Besiedlung mit dsRNA untersucht. Keines der Isolate enthielt CHV-1; nur sechs Isolate, welche alle von der Fläche mit den Stockausschlägen stammten, enthielten CHV-3. CHV-4, das natürlich in den Wäldern West-Viriginias vorkommt und in zwei der freigesetzten Isolate vorhanden war, hybridisierte mit dsRNA aus den Isolaten, die CHV-3 enthielten, was auf eine Mischinfektion hindeutet. CHV-4 hybridisierte auch mit dsRNA aus anderen Isolaten, die innerhalb und ausserhalb der behandelten Versuchsflächen gesammelt wurden. Im Gegensatz zu CHV-1 und CHV-3 war der Einfluss von CHV-4 auf den Phänotyp von C. parasitica gering. Die begrenzte Persistenz von CHV-1 und CHV-3 wird möglicherweise durch die Abnahme der C. parasitica Population als Folge des Ausbleibens der Regeneration der Edelkastanie bedingt. Erschwerend für die Edelkastanien dürfte sich zudem die Konkurrenz mit anderen Laubhölzern auswirken. [source]


TLR3-mediated signal induces proinflammatory cytokine and chemokine gene expression in astrocytes: Differential signaling mechanisms of TLR3-induced IP-10 and IL-8 gene expression

GLIA, Issue 3 2006
Chanhee Park
Abstract Viral infection is one of the leading causes of brain encephalitis and meningitis. Recently, it was reported that Toll-like receptor-3 (TLR3) induces a double-stranded RNA (dsRNA)-mediated inflammatory signal in the cells of the innate immune system, and studies suggested that dsRNA may induce inflammation in the central nervous system (CNS) by activating the CNS-resident glial cells. To explore further the connection between dsRNA and inflammation in the CNS, we have studied the effects of dsRNA stimulation in astrocytes. Our results show that the injection of polyinosinic-polycytidylic acid (poly(I:C)), a synthetic dsRNA, into the striatum of the mouse brain induces the activation of astrocytes and the expression of TNF-,, IFN-,, and IP-10. Stimulation with poly(I:C) also induces the expression of these proinflammatory genes in primary astrocytes and in CRT-MG, a human astrocyte cell line. Furthermore, our studies on the intracellular signaling pathways reveal that poly(I:C) stimulation activates I,B kinase (IKK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in CRT-MG. Pharmacological inhibitors of nuclear factor-,B (NF-,B), JNK, ERK, glycogen synthase kinase-3, (GSK-3,), and dsRNA-activated protein kinase (PKR) inhibit the expression of IL-8 and IP-10 in astrocytes, indicating that the activation of these signaling molecules is required for the TLR3-mediated chemokine gene induction. Interestingly, the inhibition of PI3 kinase suppressed the expression of IP-10, but upregulated the expression of IL-8, suggesting differential roles for PI3 kinase, depending on the target genes. These data suggest that the TLR3 expressed on astrocytes may initiate an inflammatory response upon viral infection in the CNS. © 2005 Wiley-Liss, Inc. [source]


Hepatitis C virus,infected hepatocytes extrinsically modulate dendritic cell maturation to activate T cells and natural killer cells,

HEPATOLOGY, Issue 1 2008
Takashi Ebihara
Dendritic cell maturation critically modulates antiviral immune responses, and facilitates viral clearance. Hepatitis C virus (HCV) is characterized by its high predisposition to persistent infection. Here, we examined the immune response of human monocyte-derived dendritic cells (MoDCs) to the JFH1 strain of HCV, which can efficiently replicate in cell culture. However, neither HCV RNA replication nor antigen production was detected in MoDCs inoculated with JFH1. None of the indicators of HCV interacting with MoDCs we evaluated were affected, including expression of maturation markers (CD80, 83, 86), cytokines (interleukin-6 and interferon-beta), the mixed lymphocyte reaction, and natural killer (NK) cell cytotoxicity. Strikingly, MoDCs matured by phagocytosing extrinsically-infected vesicles containing HCV-derived double-stranded RNA (dsRNA). When MoDCs were cocultured with HCV-infected apoptotic Huh7.5.1 hepatic cells, there was increased CD86 expression and interleukin-6 and interferon-beta production in MoDCs, which were characterized by the potential to activate NK cells and induce CD4+ T cells into the T helper 1 type. Lipid raft-dependent phagocytosis of HCV-infected apoptotic vesicles containing dsRNA was indispensable to MoDC maturation. Colocalization of dsRNA with Toll-like receptor 3 (TLR3) in phagosomes suggested the importance of TLR3 signaling in the MoDC response against HCV. Conclusion: The JFH1 strain does not directly stimulate MoDCs to activate T cells and NK cells, but phagocytosing HCV-infected apoptotic cells and their interaction with the TLR3 pathway in MoDCs plays a critical role in MoDC maturation and reciprocal activation of T and NK cells. (HEPATOLOGY 2008.) [source]


Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA

INSECT MOLECULAR BIOLOGY, Issue 1 2009
D. P. Walshe
Abstract Reverse genetic studies based on RNA interference (RNAi) have revolutionized analysis of gene function in most insects. However the necessity of injecting double stranded RNA (dsRNA) inevitably compromises many investigations particularly those on immunity. Additionally, injection of tsetse flies often causes significant mortality. We demonstrate, at transcript and protein level, that delivering dsRNA in the bloodmeal to Glossina morsitans morsitans is as effective as injection in knockdown of the immunoresponsive midgut-expressed gene TsetseEP. However, feeding dsRNA fails to knockdown the fat body expressed transferrin gene, 2A192, previously shown to be silenced by dsRNA injection. Mortality rates of the dsRNA fed flies were significantly reduced compared to injected flies 14 days after treatment (Fed: 10.1%± 1.8%; injected: 37.9% ± 3.6% (Mean ± SEM)). This is the first demonstration in Diptera of gene knockdown by feeding and the first example of knockdown in a blood-sucking insect by including dsRNA in the bloodmeal. [source]


Germ-line transformation and RNAi of the ladybird beetle, Harmonia axyridis

INSECT MOLECULAR BIOLOGY, Issue 4 2006
H. Kuwayama
Abstract To elucidate the molecular mechanisms underlying the tremendous diversity of insect wing colour patterns, it is imperative to identify and functionally characterize the genes involved in this developmental process. Here we report the first successful germ-line transformation using the transposable element vector piggyBac in the ladybird beetle Harmonia axyridis, which demonstrates typical genetic polymorphism in its wing colour patterns. The transformation efficiency by piggyBac was 3.7% per fertile G0. We investigated the effectiveness of RNAi in Harmonia by injecting EGFP (enhanced green fluorescent protein) dsRNA into early transgenic EGFP-expressing embryos and observed substantial reduction of EGFP fluorescence in 87.2% of hatched larvae. Application of these new genetic tools to non-model insects such as Harmonia will facilitate the broad understanding of developmental mechanisms and evolutionary processes that are inaccessible using established model systems. [source]


RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum

INSECT MOLECULAR BIOLOGY, Issue 3 2003
M. N. Aljamali
Abstract RNA interference (RNAi), a gene silencing process, 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 putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick,host interface. [source]


Specific developmental gene silencing in the honey bee using a homeobox motif

INSECT MOLECULAR BIOLOGY, Issue 6 2002
M. Beye
Abstract Manipulating the expression of genes in species that are not currently used as genetic models will provide comparative insights into the evolution of gene functions. However the experimental tools in doing so are limited in species that have not served as models for genetic studies. We have examined the effects of double stranded RNA (dsRNA) in the honey bee, an insect with considerably basic scientific interest. dsRNA derived from a 300 bp stretch of the E30 homeobox motif was injected into honey bee embryos at the anterior pole in the preblastoderm stage. We found that the dsRNA fragment successfully disrupted the protein expression of the target gene throughout the whole embryo. The disruption caused deficient phenotypes similar to known loss of function mutants of Drosophila engrailed, whereas embryos injected with nonsense dsRNA showed no abnormalities. We show that the large size of the honey bee egg (D: 0.3 mm, L: 1.6 mm) and the long preblastoderm stage (11,12 h) can be exploited to generate embryos with partial disruption of gene function, which may provide an elegant alternative to classical chimeric analyses. This is the first report of targeted disruption of gene function in the honey bee, and the results prove that the chosen target gene is a functional ortholog to engrailed in Drosophila. [source]


Induction of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white gene

INSECT MOLECULAR BIOLOGY, Issue 3 2002
G. X. Quan
Abstract Injection of double-stranded RNA (dsRNA) corresponding to the silkworm white gene (Bmwh3) into preblastoderm eggs of the wild-type silkworm induced phenotypes similar to those observed with mutants of the white egg 3 locus (10,19.6). The induced phenotypes were characterized by the presence of white eggs and translucent larval skin. Northern analysis showed that the expression of the endogenous Bmwh3 gene in the injected embryos was distinctly depressed. Furthermore, the injection of the GFP dsRNA inhibited the expression of the GFP gene from a plasmid co-injected with the dsRNA but did not depress the expression of the Bmwh3 gene. These findings demonstrate that sequence-specific RNA interference occurred in the silkworm. We conclude from the results that the RNA interference can be applied as a tool for the analysis of the gene function in the lepidopteran insects. [source]


Demography of American chestnut populations: effects of a pathogen and a hyperparasite

JOURNAL OF ECOLOGY, Issue 4 2004
ANITA L. DAVELOS
Summary 1Matrix models were used to evaluate the effect of chestnut blight infection on transition probabilities and population growth rates for American chestnuts. Disease-free, epidemic and recovering (i.e. pathogen infected with a double-stranded (ds) RNA hypovirus) populations were compared. 2Population growth rates (,) did not differ significantly over time or with disease status. However, predicted stable stage distributions differed between population types, with disease-free and recovering populations more similar to each other than either was to epidemic populations. 3Survival had the highest proportional contribution to population growth rates as revealed by elasticity analyses. However, reductions in stasis of the largest trees contributed most to reductions in population growth rate when comparing diseased with disease-free populations using LTRE. 4The presence of hypovirus reduces pathogen virulence, allowing individual American chestnut trees to increase in size. Where dsRNA has spread, chestnut populations in Michigan have attained population dynamics similar to those found in disease-free populations. 5Matrix models and life table response experiments can be used to detect important pathogen-mediated changes in the dynamics of host populations. [source]


Purification and characterization of a new reovirus from the Chinese mitten crab, Eriocheir sinensis

JOURNAL OF FISH DISEASES, Issue 12 2004
S Zhang
Abstract A new reovirus was recently isolated from a freshwater crab, the Chinese mitten crab, Eriocheir sinensis, in China. The complete viral particles are 55 nm in diameter, icosahedral, non-enveloped and have a mean buoyant density of 1.39 g cm,3 in CsCl gradient. The viral genome is composed of 12 pieces of dsRNA with an electrophoretic pattern of 3/4/2/3. This virus infects connective tissue of the gills, gut and hepatopancreas. Partial cDNA cloning and sequence analysis showed that the RNA-dependent RNA polymerase is located in the first RNA segment. From its biochemical, ultrastructural and physicochemical properties, this virus is quite different from the genus Aquareovirus (Reoviridae). It may represent a new genus of Reoviridae, different from the other crab reoviruses, P and W2. [source]


Identification of Three Strains of a Virus Associated with Cassava Plants Affected by Frogskin Disease

JOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2008
L. A. Calvert
Abstract Cassava Frogskin Disease (CFSD) can cause severe damage to cassava roots and is one of the most important diseases of cassava in Latin America. The principal objective of this study was to identify the causal agent of CFSD. Electron microscopy, viral purifications, double-stranded RNA (dsRNA) analysis, cloning, sequencing, rtPCR and hybridizations were carried out to characterize and associate a novel virus with the disease. Virus-like particles of 70 and 45 nm in diameter were found in affected cassava plants and partially purified preparations respectively. Nine species of dsRNA were associated with this disease and cDNA clones to six genomic segments were synthesized from the purified dsRNAs. The putative proteins predicted from the sequence of the cassava virus cDNA clones have similarity with the P1, P2, P3, P4, P5 and P10 proteins of Rice ragged stunt virus (RRSV). Phylogenic analysis confirmed that this virus is a member of the family Reoviridae and is most closed related to RRSV. Hybridization analyses of dsRNA identified S1, S2, S3, S4, S5 and S10 genomic segments in the CFSD-affected plants, but not in healthy controls. Additionally, 26 isolates were compared using a portion of the putative polymerase gene. The virus was detected in all 26 isolates, and they were classified into three distinct races. The association of this virus with CFSD was strengthened by the detection of this virus in diseased plants collected from different locations throughout Colombia. [source]


Melatonin decreases TLR3-mediated inflammatory factor expression via inhibition of NF-,B activation in respiratory syncytial virus-infected RAW264.7 macrophages

JOURNAL OF PINEAL RESEARCH, Issue 1 2008
Sheng-Hai Huang
Abstract:, Double-stranded (ds) RNA has been identified as a ligand for Toll-like receptor 3 (TLR3). Respiratory syncytial virus (RSV), a single-stranded RNA virus and a major respiratory pathogen and pneumovirus in human infants pathogenesis of which relies on early inflammatory and immune events of the host in response to RSV, could be recognized by TLR3 sensing viral dsRNA produced during replication. The downstream signaling pathway from TLR3 leads to activation of IFN regulatory factor (IRF)-3 and/or NF-,B and subsequent expression of numerous proinflammatory factors. Melatonin (MT) is an effective regulator of the immune system. To determine the molecular mechanisms responsible for the suppressive effect of MT on RSV infection, we analyzed signaling molecules involved in the TLR3-mediated activation of inflammatory factors in macrophages infected with RSV and the modulatory role of MT on these mediators. We report that RSV infection of RAW264.7 macrophages time-dependently stimulate the rapid activation of TLR3 and NF-,B, as well as subsequent NF-,B-dependent gene expression such as those encoding TNF-, and inducible nitric oxide synthase. Moreover, we demonstrate that MT decreased TLR3-mediated downstream gene expression in RSV-infected macrophages in a dose- and time-dependent manner, and that MT inhibition of NF-,B activity seemed to be the key event required to explain the reduction in inflammatory gene expression caused by MT. But MT did not influence TLR3 at either the protein or mRNA level or MyD88 transcription. These results could be related to the beneficial immunoregulatory role of MT in RSV-infected macrophages and address the possible therapeutic potential of this indoleamine in human RSV diseases. [source]


Ethanol Promotes Thiamine Deficiency-Induced Neuronal Death: Involvement of Double-Stranded RNA-activated Protein Kinase

ALCOHOLISM, Issue 6 2009
Zun-Ji Ke
Background:, Heavy alcohol consumption causes cerebellar degeneration, and the underlying mechanism is unclear. Chronic alcoholism is usually associated with thiamine deficiency (TD) which is known to induce selective neurodegeneration in the brain. However, the role of TD in alcohol-induced cerebellar degeneration remains to be elucidated. The double-stranded RNA-activated protein kinase (PKR) is a potent antiviral protein. Viral infection or binding to dsRNA causes PKR autophosphorylation and subsequent phosphorylation of the ,-subunit of eukaryotic translation factor-2,, leading to inhibition of translation or apoptosis. PKR can also be activated by cellular stresses. Methods:, In this study, we used an in vitro model, cultured cerebellar granule neurons (CGNs), to investigate the interaction between TD and ethanol and evaluate the contribution of their interaction to neuronal loss. TD was induced by treatment with amprolium in association with ethanol. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay. PKR expression/phosphorylation and subcellular distribution was analyzed with immunoblotting and immunocytochemistry. Results:, Thiamine deficiency caused death of CGNs but ethanol did not. However, TD plus ethanol induced a much greater cell loss than TD alone. TD-induced PKR phosphorylation and ethanol exposure significantly promoted TD-induced PKR phosphorylation as well as its nuclear translocation. A selective PKR inhibitor not only protected CGNs against TD toxicity, but also abolished ethanol potentiation of TD-induced loss of CGNs. Conclusions:, Ethanol promoted TD-induced PKR activation and neuronal death. PKR may be a convergent protein that mediates the interaction between TD and ethanol. [source]


A viral PAMP double-stranded RNA induces allergen-specific Th17 cell response in the airways which is dependent on VEGF and IL-6

ALLERGY, Issue 10 2010
J.-P. Choi
To cite this article: Choi J-P, Kim Y-S, Tae Y-M, Choi E-J, Hong B-S, Jeon SG, Gho YS, Zhu Z, Kim Y-K. A viral PAMP double-stranded RNA induces allergen-specific Th17 cell response in the airways which is dependent on VEGF and IL-6. Allergy 2010; 65: 1322,1330. Abstract Background:, Innate immune response by a viral pathogen-associated molecular pattern dsRNA modulates the subsequent development of adaptive immune responses. Although virus-associated asthma is characterized by noneosinophilic inflammation, the role of Th17 cell response in the development of virus-associated asthma is still unknown. Objective:, To evaluate the role of the Th17 cell response and its underlying polarizing mechanisms in the development of an experimental virus-associated asthma. Methods:, An experimental virus-associated asthma was created via airway sensitization with ovalbumin (OVA, 75 ,g) and a low (0.1 ,g) or a high (10 ,g) doses of synthetic dsRNA [polyinosine,polycytidylic acid; poly(I:C)]. Transgenic (IL-17-, IL-6-deficient mice) and pharmacologic [a vascular endothelial growth factor receptor (VEGFR) inhibitor] approaches were used to evaluate the roles of Th17 cell responses. Results:, After cosensitization with OVA and low-dose poly(I:C), but not with high-dose poly(I:C), inflammation scores after allergen challenge were lower in IL-17-deficient mice than in wild-type (WT) mice. Moreover, inflammation enhanced by low-dose poly(I:C), but not by high-dose poly(I:C), was impaired in IL-6-deficient mice; this phenotype was accompanied by the down-regulation of IL-17 production from T cells from both lymph nodes and lung tissues. Airway exposure of low-dose poly(I:C) enhanced the production of VEGF and IL-6, and the production of IL-6 was blocked by treatment with a VEGFR inhibitor (SU5416). Moreover, the allergen-specific Th17 cell response and subsequent inflammation in the low-dose poly(I:C) model were impaired by the VEGFR inhibitor treatment during sensitization. Conclusions:, Airway exposure of low-level dsRNA induces an allergen-specific Th17 cell response, which is mainly dependent on VEGF and IL-6. [source]


Opposing roles of IL-17A and IL-25 in the regulation of TSLP production in human nasal epithelial cells

ALLERGY, Issue 5 2010
G. Xu
To cite this article: Xu G, Zhang L, Wang DY, Xu R, Liu Z, Han DM, Wang XD, Zuo KJ, Li HB. Opposing roles of IL-17A and IL-25 in the regulation of TSLP production in human nasal epithelial cells. Allergy 2010; 65: 581,589. Abstract Background:, The importance of IL-17A, IL-17F, and IL-25 in allergic rhinitis (AR), as well as their possible role in regulation on thymic stromal lymphopoietin (TSLP) production in nasal epithelial cells, is not well understood. Objective:, To determine the possible regulation of IL-17A, IL-17F, and IL-25 on TSLP production in the initiation of allergic responses. Methods:, The levels of IL-17A, IL-17F, IL-25, and TSLP in nasal lavages of patients with AR were measured using an enzyme-linked immunosorbent assay (ELISA) and compared with that in normal controls. Then, primary human nasal epithelial cells (HNECs) were stimulated with dsRNA (0,75 ,g/ml), as well as IL-17A (100 ng/ml), IL-17F (100 ng/ml), and IL-25(100 ng/ml). The mRNA expression of IL-17A, IL-17F, IL-25, TSLP, as well as the chemokines CCL20, IL-8, and eotaxin was analyzed using quantitative real-time PCR, and their protein levels in the supernatants of cultured HNECs were determined by ELISA. Results:, Both TSLP and IL-17 cytokines are significantly elevated in patients with AR. dsRNA was found to increase the production of IL-17F, IL-25, TSLP, CCL20, and IL-8 in HNECs. Furthermore, IL-25 significantly enhanced dsRNA-induced TSLP production in primary HNECs and was dominant to the inhibitory effect of IL-17A on TSLP regulation. Conclusions:, Our study provides the first evidence that both IL-17F and IL-25 can be induced by dsRNA in HNECs. Despite of the opposing effects of IL-17A and IL-25 on TSLP regulation in HNECs, IL-25 was dominant to IL-17A, providing a plausible explanation for the simultaneous upregulation of IL-17 cytokines and TSLP in patients with AR. [source]


Recent progress in the development of RNA interference for plant parasitic nematodes

MOLECULAR PLANT PATHOLOGY, Issue 5 2007
CATHERINE J. LILLEY
SUMMARY RNA interference (RNAi), first described for Caenorhabditis elegans, has emerged as a powerful gene silencing tool for investigating gene function in a range of organisms. Recent studies have described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when preparasitic juvenile nematodes take up double-stranded (ds)RNA that elicits a systemic RNAi response. Important developments over the last year have shown that in planta expression of a dsRNA targeting a nematode gene can successfully induce silencing in parasitizing nematodes. When the targeted gene has an essential function, a resistance effect is observed paving the way for the potential use of RNAi technology to control plant parasitic nematodes. [source]


Rust of flax and linseed caused by Melampsora lini

MOLECULAR PLANT PATHOLOGY, Issue 4 2007
GREGORY J. LAWRENCE
SUMMARY Melampsora lini, while of economic importance as the causal agent of rust disease of flax and linseed, has for several decades been the ,model' rust species with respect to genetic studies of avirulence/virulence. Studies by Harold Flor demonstrated that single pairs of allelic genes determine the avirulence/virulence phenotype on host lines with particular resistance genes and led him to propose his famous ,gene-for-gene' hypothesis. Flor's inheritance studies, together with those subsequently carried out by others, also revealed that, in some cases, an inhibitor gene pair and an avirulence/virulence gene pair interact to determine the infection outcome on host lines with particular resistance genes. Recently, avirulence/virulence genes at four loci, AvrL567, AvrM, AvrP4 and AvrP/AvrP123, have been cloned. All encode novel, small, secreted proteins that are recognized inside plant cells. Yeast two-hybrid studies have shown that the AvrL567 proteins interact directly with the resistance gene protein. The molecular basis of Flor's gene-for-gene relationship has now been elucidated for six interacting gene pairs: those involving resistance genes L5, L6, L7, M, P and P2, where both the resistance gene and the corresponding avirulence gene have been cloned. In other inheritance studies it has been shown that M. lini does not possess a (+) and (,) mating system, but may possess a two factor system. Double-stranded (ds) RNA molecules occur in many strains of M. lini: examination of the progeny of one strain that possesses 11 dsRNA molecules revealed that they fall into three transmission units, designated L, A and B. The L unit consists of a single large dsRNA of 5.2 kbp while the A and B units each consist of five dsRNAs in the size range 1.1,2.8 kbp. The three units have different sexual and asexual transmission characteristics. The L unit is encapsidated in a virus-like particle, whereas the other units are not encapsidated. The population and coevolutionary aspects of M. lini on a wild, native Australian host species, Linum marginale, have been extensively investigated. A recent molecular analysis revealed that the M. lini isolates from L. marginale fall into two distinct lineages, one of which is apparently hybrid between two diverse genomes. Isolates in this lineage are largely fixed for heterozygosity, which suggests that sexual recombination does not occur in this lineage. [source]


Oocyte-selective expression of MT transposon-like element, clone MTi7 and its role in oocyte maturation and embryo development

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004
Chang-Eun Park
Abstract Previously, we found MT transposon-like element, clone MTi7 (MTi7) is highly expressed in the mouse ovary. Here, we show that the MTi7 is expressed in the oocyte from the primordial to the preovulatory follicles. For RNA interference (RNAi), double stranded RNAs (dsRNAs) were prepared for MTi7 and c-mos, a control gene with known functions. Each dsRNA was microinjected into germinal vesicle (GV) stage oocytes or zygotes with pronuclei (PN), after which developmental changes, mRNA expression, and nuclear and microtubular organization were analyzed. We found a 43.4,53% GV arrest in the microinjected oocytes with a concomitant decrease in targeted mRNA expression. In MTi7 dsRNA-injected early and late PN zygotes, a 92.9% 1-cell arrest and 76.9% 2-cell arrest were observed, respectively. This is the first report of an oocyte-selective expression of MTi7 mRNA, and our results strongly suggest that MTi7 involved in the nuclear membrane breakdown during oocyte maturation and embryo development. Mol. Reprod. Dev. 69: 365,374, 2004. © 2004 Wiley-Liss, Inc. [source]


Development of a reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Amasya cherry disease

PLANT PATHOLOGY, Issue 6 2007
Z. Kozlakidis
A reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed to detect Amasya cherry disease (ACD) in naturally infected sweet cherry (Prunus avium) leaves sampled from Turkey. The procedure was based on detection of the presence of a mycoviral-like double-stranded RNA (dsRNA) of 5·3 kbp always found in association with ACD, which is probably caused by a fungus. Specific primers were designed to amplify a fragment of the diagnostic dsRNA. The method will improve routine diagnosis of ACD in Prunus spp. [source]


Study of PfMyb1 Transcription Factor Regulation Network during Plasmodium falciparum Erythrocytic Cycle

THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005
M. GISSOT
During the complex life cycle of Plasmodium falciparum, the regulation of events that occur during the erythrocytic cycle, such as proliferation and differentiation, implies a fine control of transcriptional activities governing the expression profiles of each gene. However, transcriptional regulation and notably its actors, transcription factors and regulation motifs, are poorly described in Plasmodium. In order to decipher the mechanisms implicated in transcriptional regulation, we studied a transcription factor belonging to the trytophan family and showed that the PfMyb1 protein contained in nuclear extracts has a specific DNA binding activity. We took advantage of long pfmyb1 double stranded RNA (dsRNA) to inactivate the cognate messenger and understand the role of PfMyb1 during the erythrocytic cycle. Culture treated with pfmyb1 dsRNA exhibited a 40% growth inhibition and mortality during trophozoite to schizont transition when compared to either untreated control or culture treated with unrelated long dsRNA. We have further demonstrated that pfmyb1 transcript and protein decreased up to 80% in treated trophozoite culture at the time of pfmyb1 expression peak. Thus, we investigated the effect of this partial loss of transcript and protein using a thematic DNA microarray containing PCR products, representative of P. falciparum genes involved in cell cycle and transcriptional regulation. SAM software enabled us to identify several genes over and under-expressed, potentially directly or indirectly regulated by PfMyb1. These alterations of expression were verified by qPCR and Western blotting. We are currently working on the promoters of those genes to decode determinants of gene regulation by Pfmyb1. [source]


RNA interference for antiviral therapy

THE JOURNAL OF GENE MEDICINE, Issue 8 2006
Mali Ketzinel-Gilad
Abstract 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. Copyright © 2006 John Wiley & Sons, Ltd. [source]


Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells

THE JOURNAL OF GENE MEDICINE, Issue 7 2004
Makoto Miyagishi
Abstract Background RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells. Methods We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors. Results As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five. Conclusion The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi. Copyright © 2004 John Wiley & Sons, Ltd. [source]


OsRecQ1, a QDE-3 homologue in rice, is required for RNA silencing induced by particle bombardment for inverted repeat DNA, but not for double-stranded RNA

THE PLANT JOURNAL, Issue 2 2008
Hui Chen
Summary Based on the nucleotide sequence of QDE-3 in Neurospora crassa, which is involved in RNA silencing, rice (Oryza sativa) mutant lines disrupted by the insertion of the rice retrotransposonTos17 were selected. Homozygous individuals from the M1 and M2 generations were screened and used for further analyses. The expression of the gene was not detected in leaves or calli of the mutant lines, in contrast to the wild type (WT). Induction of RNA silencing by particle bombardment was performed to investigate any effects of the OsRecQ1 gene on RNA silencing with silencing inducers of the GFP (green fluorescence protein)/GUS (, -glucuronidase) gene in the mutant lines. The results showed that OsRecQ1 is required for RNA silencing induced by particle bombardment for inverted-repeat DNA, but not for double-stranded RNA (dsRNA). The levels of transcripts from inverted-repeat DNA were much lower in the mutant lines than those in the WT. Furthermore, no effects were observed in the accumulation of endogenous microRNAs (miR171 and miR156) and the production of the short interspersed nuclear element retroelement by small interfering RNA. On the basis of these results, we propose that OsRecQ1 may participate in the process that allows inverted repeat DNA to be transcribed into dsRNA, which can trigger RNA silencing. [source]