Suppression Of Transgene Expression Research Articles

Elevated cholecystokininergic tone constitutes an important molecular/neuronal mechanism for the expression of anxiety in the mouse

Cholecystokinin (CCK), one of the most abundant neuropeptides in the brain, plays an important role in anxiogenesis through the activation of CCK receptor-2 (CCKR-2). Accumulating evidence, however, has suggested this role depends on endogenous CCKergic "tone," which is largely determined by the expression level of the CCKR-2. Using the tTA/tetO-inducible transgenic (tg) approach, we show here that overexpression of the CCKR-2 in neurons of the forebrain significantly increases CCKR-2 binding capacity in tg mice compared with their littermate controls. Interestingly, these tg mice consistently exhibit increased fear responses, which are generally interpreted as anxiety-like behaviors in the rodent, in a battery of behavioral tests, which represented conflict situations or delivered stress to the subjects. The inhibition of transgene expression with doxycycline treatment completely diminished both increased receptor-binding activity and all behavioral phenotypes. Furthermore, treatment of tg mice with diazepam significantly attenuated these anxiety-like behaviors. Our results directly demonstrate that the elevated CCKergic tone via overexpression of the CCKR-2 in the brain may constitute an underlying molecular/neuronal mechanism for the expression of anxiety. In addition, our study has validated a robust genetic anxiety model in the mouse in terms of their face, constructive, and predictive validity.

803. Endogenous microRNA Regulation Suppresses Transgene Expression in Hematopoietic Lineages and Enables Stable Gene Transfer

One of the major barriers to stable gene transfer is the development of anti-transgene immunity. In studies of gene therapy for inherited diseases, such as the hemophilias, successful outcome has been precluded by the development of immune responses against the vector and transgene product. Several factors contribute to the induction of an immune response following gene transfer. Chief amongst these factors is the expression of the transgene product within antigen presenting cells (APCs). Tissue-specific promoters (TSPs) can be used to prevent expression within APCs.

100. Inducible and Reversible Transgene Expression in Human Stem Cells Stably Transduced by Novel Lentiviral Vectors

It is ideal, and quite often necessary, to have regulated or inducible transgene expression in stem cell-based gene therapies or basic studies, in which stage-specific or lineage-specific gene expression is crucial in analyzing progeny cells after stable gene transfer to the ancestral stem cells. We have previously shown that self-inactivating (SIN) lentiviral vectors confer promoter-specific transgene expression. In this study, we used the tetracycline repressor (tetR) that is fused with the KRAB transcriptional suppression domain (tTS) to specifically suppress transgene expression from stably transduced human cells. The transgene expression is controlled by a regular promoter adjacent to a high affinity-binding site (tetO7) of tTS. After optimizing a lentiviral vector containing a strong ubiquitous promoter such as EF1a and UBC and the location of the tetO7 site in the vector, we achieved better inducible transgene expression than previous lentiviral system containing the |[ldquo]|tet-off|[rdquo]| or |[ldquo]|tet-on|[rdquo]| system. In the new system, the transgene transcription from a regular promoter such as EF1a or UBC is suppressed by the tTS bound to the nearby tetO7 site in the absence of a tet analog doxycycline (Dox). In the presence of Dox, however, the tTS binding is off from the vector DNA and the regular transcription is restored. Thus the new system (dubbed as tet-SUPER) simply adds an extra level of regulation on transgene transcription, regardless which type of promoter is used. We tested the tet-SUPER system in human CD34- positive multipotent hematopoietic progenitor cell line TF1 and human embryonic stem (hES) cell lines H1 and I-6, using a dual-gene vector containing the GFP reporter (titers remain at 10-20 million transducing units per ml). In both human cell systems, we observed a tight regulation (suppression) in the un-induced or |[ldquo]|off|[rdquo]| state when Dox is absent. However, the suppression is reversible even after long-term suppression in the absence of Dox. The specific suppression was abolished and transgene expression was restored at a very low level of Dox (5 ng/ml). The presence of the tet-SUPER system did not alter unique properties of hES cells. In addition, the tet-SUPER lentiviral vector also conferred inducible transgene expression of the human PIG-A gene in a mutated human cell line that is deficient in the PIG-A gene and thus GPI-anchored proteins on cell surface. Together with recent papers published by Dr. Trono and others, we show that it is now possible to achieve inducible and reversible transgene expression in essentially any mammalian cell types.

Cellular uptake and activation characteristics of naked plasmid DNA and its cationic liposome complex in human macrophages

Plasmid DNA (pDNA) is an important macromolecular therapeutic agent suitable for DNA-based therapies, such as non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs abundant in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits transgene expression, while modulating immunological consequences following vaccination. We studied cellular uptake and activation characteristics of naked pDNA and its cationic liposome complex in human macrophage-like cells. The present study has demonstrated that naked pDNA was recognized by human macrophage-like cells via specific mechanisms for polyanions. Moreover, it has shown that pDNA complexed with cationic liposomes activates human macrophage-like cells to induce the production of tumor necrosis factor-α (TNF-α) in a CpG motif-independent manner, while any types of naked DNA could not induce TNF-α production from these cells, regardless of the presence of CpG motifs in pDNA or oligonucleotide (ODN). These findings form an important basis for DNA-based therapies including gene therapy and DNA vaccination.

RNA Interference-Based Gene Silencing in Mice: The Development of a Novel Therapeutical Strategy

RNAi (RNA interference) was originally detected in Caenorhabditis elegans as biological response to exogenous double-stranded RNA (dsRNA), which induces very effective sequence-specific silencing of gene expression. Further investigations revealed that RNAi can occur in many eukaryotic species. Increasing understanding of the biochemical components of RNAi indicates the existence of a conserved machinery for dsRNA-induced gene silencing that acts in two steps. In the first step, an RNase III family nuclease called Dicer processes the dsRNA to small interfering RNAs (siRNAs) 21-23 nt in length. These siRNAs enter a multimeric nuclease complex that identifies target mRNAs through their homology to siRNAs and induce destruction of the corresponding mRNAs. Since RNAi has become an excellent strategy for gene silencing, it is tempting to apply this technology to 'knock-down' gene expression in living animals. The generation of transgenic mice from embryonic stem cells expressing small hairpin RNAs (shRNAs) has provided evidence for in vivo application of RNAi. Furthermore, different experimental strategies have been developed to analyze the influence of chemically synthesized siRNAs and of vector-based shRNAs on the expression of different transgenes and endogenous genes in vivo. Recent studies describe the in vivo delivery of siRNAs to inhibit transgene expression in certain organs of adult mice, predominately murine liver. Strategies for the inhibition of cellular proliferation by systemic treatment of tumor-bearing animals with siRNAs are beginning to emerge. They are of utmost interest for systemic diseases such as cancer. In addition, several groups have shown that RNAi can also be used to block the infectivity or suppress the replication of different RNA viruses relevant to human diseases including human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV). In summary, multiple lines of evidence indicate that RNAi seems to become a powerful tool for the fight against undesirable gene expression in human diseases.

Type I Interferons potently suppress gene expression following gene delivery using liposome–DNA complexes

Gene delivery by intravenous injection of cationic liposome-DNA complexes (LDC) can generate efficient transgene expression in the lungs and other organs, but the duration of expression is typically short. Previous studies have suggested a major role for interferon-gamma (IFN-gamma) and TNF in this process. However, plasmid DNA is also capable of eliciting production of type I IFNs. Therefore, we assessed the ability of LDC to elicit production of type I IFNs in vivo and assessed the effects of type I IFNs on suppression of transgene expression following in vivo gene delivery with LDC. Injection of LDC was found to induce production of high levels of both IFN-alpha and IFN-beta in vivo. Moreover, the levels of transgene expression following in vivo gene delivery were markedly increased in mice lacking functional type I IFN receptor genes, compared to wild-type mice or mice lacking IFN-gamma or TNF receptors. Addition of recombinant IFN-alpha and IFN-beta inhibited transgene expression by in vitro-transfected endothelial cells, and incubation of macrophages with LDC in vitro triggered production of both IFN-alpha and IFN-beta. Therefore, type I IFNs appear to play a key role in suppressing transgene expression in vivo following systemic nonviral gene delivery using LDC.

WAVE2 Signaling Mediates Invasion of Polarized Epithelial Cells by Salmonella typhimurium

The bacterial pathogen Salmonella penetrates the intestinal epithelium by inducing its own phagocytosis into epithelial cells. The dramatic reorganization of the actin cytoskeleton required for internalization is driven by bacterial manipulation of host signaling pathways, including activation of the Rho family GTPase Rac1 and subsequent activation of the Arp2/3 complex. However, the mechanisms linking these two events remain poorly understood. Rac1 is thought to promote activation of the Arp2/3 complex through its interaction with suppressor of cAMP receptor/WASP family verprolin-homologous (SCAR/WAVE) family proteins, but this interaction is apparently indirect. Two different Rac1 effectors have been shown to bind WAVE2: IRSp53, the SH3 domain of which binds the WAVE2 proline-rich domain, and PIR121/Sra-1, which forms a pentameric complex containing WAVE, Abi1, Nap1, and HSPC300. However, the extent to which each of these complexes contributes to Arp2/3 complex activation in the context of Salmonella infection is unclear. Here, we show that WAVE2 is necessary for efficient invasion of epithelial cells by Salmonella typhimurium. We found that although Salmonella infection strongly promotes the formation of an IRSp53/WAVE2 complex, IRSp53 is not necessary for bacterial internalization. In contrast, disruption of the PIR121/Nap1/Abi1/WAVE2/HSPC300 complex potently inhibits bacterial uptake. These results indicate that WAVE2 is an important component in signaling pathways leading to Salmonella invasion. Although infection leads to the formation of an IRSp53/WAVE2 complex, it is the association of WAVE2 with the Abi1/Nap1/PIR121/HSPC300 complex that regulates bacterial internalization.

Development of a murine prostatespecific 2-in-1 inducible bicistronic expression vector

Transgenic research often suffers from the lack of strong tissue specific promoters and the lack of suitable antibodies for transgene detection. We have now constructed a novel prostate specific 2-in-1 Tetracycline-off (Tet-off), bicistronic expression vector, designated PbTetOIE, for transgenic research. The vector allows potent induction as well as inducible suppression of transgene expression in the prostate epithelial cells, and also allows the detection of transgene expression at the cellular level through the detection of the internal enhanced green fluorescent protein (EGFP) downstream of the transgene.

946. CD8+ T Cells Are Partially Mediate the Immune-Mediated Silencing of Adenoviral Vector-Encoded Transgene Expression in the Rat Brain

First generation adenoviral vectors (RAd) transfer genes into the brain with high efficiency, and provide high levels, long term expression, and therapeutic efficacy in models of brain tumors and neurodegeneration, in the absence of priming of an anti-adenoviral immune response. Systemic immunization against RAd, however, induces a cellular adaptive immune response which completely eliminates the expression of the transgene from the brain, and causes a long-term inflammatory response. To elucidate the mechanisms by which the immune response clears transgene expression from the brain, we examined, following the delivery of RAds into the striatum, and systemic immune priming, whether the immune mediated inhibition of transgene expression causes brain neurotoxicity, reduces the levels of transgene transcripts, reduces vector genome copy numbers, and is mediated by CD8+ T cells. Our experiments provided the following results: (1) at 7 and 14 days post-immunization there was an infiltration of lymphocytes and macrophages surrounding transduced cells; (2) macrophages containing remains of transduced cells could be identified indicating the existence of cytotoxicity of transduced cells; (3) there was neuronal loss in the substantia nigra, but not in the striatum; (4) transgene mRNA levels were reduced by more than 90%, but genome copy numbers were only reduced by 10-20%; (5) the depletion of CD8+ T cells using the monoclonal antibody OX8, reduced circulating CD8+ T cells by >90% and partially protected against the immunization-induced silencing of transgene expression. In summary, our data indicate that immune-mediated silencing of RAd-encoded transgene expression in the brain occurs through a combination of non-cytolytic, and, also cytotoxic mechanisms. The percentage contribution of each mechanism is currently being evaluated.

557. Unraveling the Molecular Mechanisms Responsible for Episomal Transgene Silencing In Vivo

We previously demonstrated that plasmid bacterial DNA sequences transcriptionally inactivate transgene expression while minicircle DNA vector free of plasmid bacterial backbone is persistently active in vivo. To further elucidate the underlying molecular mechanism, we studied the conditions required for the plasmid bacterial backbone to suppress transgene expression. We found that when circular or linearized minicircle encoding the RSV.hAAT.bpA expression cassette co-injected with circular or linear pBlueScript to mouse liver via hydrodynamic technique, transgene silencing occurred when 2 linear DNAs were infused together, but not when either one or both DNA elements were in circular form, or if the linear expression cassette was injected alone. Southern analyses confirmed that linear DNA but not circular DNA molecules formed random concatemers. This further confirmed our earlier findings that transcriptional silencing occurred only when the bacterial plasmid sequences were covalently attached to the expression cassette, and bacterium-derived plasmid DNA molecules, either in circular or in concatemer form, present in the same cells will not silence DNA expression cassettes in trans-. Furthermore, we found that after injection of a mixture of linear DNA expression cassettes and plasmid bacterial DNAs into normal or SCID mice, silencing was observed in both strains of mouse. These observations strongly suggest that toxic, and probably immunogenic, signaling from plasmid derived DNAs is not responsible for DNA silencing. To determine if DNA methylation was associated with silencing, both transgene and bacterial backbone DNAs were generated from the cytosine methylation-deficient bacterial strain, SCS110. Southern analyses using methylation sensitive restriction enzymes, showed no in vivo cytosine methylation in the CpG motif. This suggests that DNA methylation is not responsible for transcriptional silencing. Interestingly, transgene silencing was alleviated when the RSV.hAAT.bpA expression cassette was flanked with 2 tandem copies of the chicken SH4 insulator at each end. Taken together our results support our hypothesis that the transcriptional suppression is due to differential nucleosome formation and/or histone modifications that are formed in the presence of plasmid bacterial DNA sequences, and then spread to suppress the transgene promoter in cis-.

Regulation of α-Smooth Muscle Actin Expression in Granulation Tissue Myofibroblasts Is Dependent on the Intronic CArG Element and the Transforming Growth Factor-β1 Control Element

Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of alpha-smooth muscle actin (alpha-SMA); however, little is known regarding molecular mechanisms that control activation of alpha-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for alpha-SMA expression in myofibroblasts in vivo and to determine whether activation of alpha-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)6GG] and a transforming growth factor-beta1 control element (TCE) that are required for alpha-SMA expression in smooth muscle cells. A Lac Z transgene construct from -2600 through the first intron was expressed in myofibroblasts within granulation tissue of cutaneous wounds in a pattern that closely mimicked endogenous alpha-SMA expression. Mutation of either the intronic CArG element or the TCE completely inhibited transgene expression in myofibroblasts in granulation tissue and responsiveness to transforming growth factor-beta1 in cultured transgenic fibroblasts. These same elements were also critical in regulating alpha-SMA expression during skeletal muscle repair but not during skeletal muscle development. Taken together, these results provide the first in vivo evidence for the importance of the intronic CArG and TCE cis-elements in the regulation of alpha-SMA expression in myofibroblasts in granulation tissue.

Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery

Owing to their capacity to induce strong, sequence-specific, gene silencing in cells, short interfering RNAs (siRNAs) represent new potential therapeutic tools. This development requires, however, new safe and efficient in vivo siRNA delivery methods. In the present technical report, we show that electrically mediated siRNA transfer can suppress transgene expression in adult mice muscles. Using electropulsation for siRNA delivery opens the way for a targeted gene silencing on a broad range of tissues. Clinical applications of electropulsation for delivery of other classes of molecules are under trials. We reported that gene silencing was efficiently obtained in vivo in an adult mammal (mouse) with chemically synthesized siRNA after its electrical delivery. The associated gene silencing was followed on the same animal and lasted at least 11 days. Gene silencing was obtained in muscles not only on young adult mice but also on much older animals. No tissue damages were detected under our electrical conditions. Therefore, this method should provide an efficient approach for a localized delivery of siRNAs in various tissues and organs.

CBP Histone Acetyltransferase Activity Is a Critical Component of Memory Consolidation

The stabilization of learned information into long-term memories requires new gene expression. CREB binding protein (CBP) is a coactivator of transcription that can be independently regulated in neurons. CBP functions both as a platform for recruiting other required components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. To dissect the chromatin remodeling versus platform function of CBP or the developmental versus adult role of this gene, we generated transgenic mice that express CBP in which HAT activity is eliminated. Acquisition of new information and short-term memory is spared in these mice, while the stabilization of short-term memory into long-term memory is impaired. The behavioral phenotype is due to an acute requirement for CBP HAT activity in the adult as it is rescued by both suppression of transgene expression or by administration of the histone deacetylase inhibitor Trichostatin A (TSA) in adult animals.

459. Immune System Regulation of Transgene Expression in the Brain 1: Systemically Activated Immune Responses Silence Adenoviral Vector-Encoded Transgene Expression in the Brain through Both Non-Cytolytic, and Cytotoxic Mechanisms

Top of pageAbstract First generation adenoviral vectors (RAd) transfer genes into the brain with high efficiency, and provide high levels and long term expression in the absence of priming of an anti-adenoviral immune response. Systemic immunization to the RAd induces a cellular adaptive immune response which eliminates the expression of the transgene from the brain, and causes a chronic inflammatory response. In order to elucidate the mechanisms by which the immune response clears transgene expression from the brain, we asked whether, following the delivery of RAds into the striatum, and systemic immune priming, the immune mediated inhibition of transgene expression: (i) reduces the levels of transgene transcripts, vector genome copy numbers, or both; (ii) was transgene dependent; (iii) was promoter dependent; and (iv) caused brain neurotoxicity. The answer to these questions can indicate the mechanisms by which the adaptive immune response controls transgene expression from viral vectors in the brain. Our experiments provided the following results: (1) Immune-mediated inhibition was transgene independent (both expression of b-gal or HSV1-TK were eliminated); (2) the immune system reduced the levels of mRNA encoding for the transgene by more than 90%, but genome copy numbers were only reduced by 10–20%; (3) activated lymphocytes entering the brain were equally efficient in inhibiting expression from viral murine-CMV, human-CMV, RSV promoters, the neuronal synapsin promoter, and the housekeeping b-actin promoter; (4) localized and statistically significant neurotoxicity was encountered in the substantia nigra, the area of the brain transduced following injection of viral vectors into the striatum. In summary, our data indicate that immune-mediated silencing of RAd-encoded transgene expression in the brain is transgene independent, promoter independent, and acts mainly by reducing the number of mRNA. Importantly, we were able to uncover a localized, but statistically significant reduction in the number of transduced neurons. This strongly suggests that activated lymphocytes exert transgene silencing in the brain through a combination of non-cytolytic, and to a lesser extent, also cytotoxic mechanisms.

RNA interference in crop plants.

RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in various organisms. Although RNAi was first discovered in worms, related phenomena such as post-transcriptional gene silencing and coat protein mediated protection from viral infection had been observed in plants prior to this. In plants, RNAi is often achieved through transgenes that produce hairpin RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. It also offers advantages over mutation-based reverse genetics in its ability to suppress transgene expression in multigene families in a regulated manner.

Effect of adeno-associated virus-specific immunoglobulin G in human amniotic fluid on gene transfer.

Intra-amniotic administration of adeno-associated virus (AAV) vector may be an effective way to deliver gene therapy for treatment of congenital pulmonary and intestinal disorders. In an effort to understand potential barriers to intra-amniotic gene therapy better, we determined whether human amniotic fluid (AF) could act as an inhibitor of AAV2-mediated gene transfer. AF samples were obtained from 21 different human pregnancies during routine amniocentesis at 16-20 weeks of gestation. An immortalized fetal human tracheal epithelial cell line (FHTE) was infected with AAV2 containing a luciferase reporter gene driven by the SV40 promoter in the presence and absence of each AF sample. Inhibition of transgene expression was observed in 8 (38%) of the AF samples (inhibitory AF) and resulted in luciferase levels of only 1.4% +/- 0.6% of those obtained with infection in normal media. Infections in 13 samples (62%) resulted in transgene expression comparable or in excess of infection in media alone (noninhibitory AF). Removal of immunoglobulin G (IgG) from inhibitory AF samples with Protein A returned luciferase expression to control levels (119% +/- 37% of control), suggesting the possible presence of inhibiting antibody. Eleven of the AF samples were evaluated by enzyme-linked immunosorbent assay (ELISA) for specific anti-AAV antibodies. All noninhibitory AF samples were negative (titers of < 1:20; n = 3), and 6 of the 8 inhibitory samples contained specific anti-AAV antibodies at titers ranging from 1:40 to 1:160. These studies demonstrate that AF from some individuals contains AAV-specific IgG that can inhibit gene transfer.

How to make tetracycline-regulated transgene expression go on and off

Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3 h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.

Differential Top10 promoter regulation by six tetracycline analogues in plant cells.

The effects of five tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline, on Top10 promoter activity in NT1 tobacco tissue culture cells have been analysed. The concentration that repressed Top10 promoter activity, the level of transgene repression and the kinetics of transgene de-repression were determined for each analogue, and could not be predicted from in vitro binding affinity to the tetracycline repressor or from comparison with animal cells. Doxycycline had the most potent effect on the Top10 promoter and completely inhibited transgene expression at 4 nmol l(-1). Tetracycline was the most versatile of the analogues tested; tetracycline inhibited the Top10 promoter at 10 nmol l(-1) and was easily washed out to restore Top10-driven expression in 12-24 h. A study was also made of the suitability for plant research of a novel tetracycline analogue, GR33076X. In animal cells, GR33076X de-repressed Top10 promoter activity in the presence of inhibitory concentrations of anhydrotetracycline. In NT1, it is shown that GR 33076X can antagonize repression of the Top10 promoter in the presence of tetracycline, but not of anhydrotetracycline or of doxycycline. Different tetracycline analogues can therefore be used to regulate the Top10 promoter in plant cells and this property may be exploited in planning an optimum course of transgene regulation.

Gene transfer approaches to the healing of bone and cartilage.

Gene transfer approaches to the healing of bone and cartilage.

Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference.

Mosquitoes transmit numerous viral pathogens to humans including dengue virus which affects approximately 50 million individuals per year. Inhibition of viral gene expression within an insect host could be used to block virus replication and subsequent transmission of the pathogen to humans. A naturally occurring gene silencing mechanism triggered by double-stranded RNA (dsRNA), RNA interference (RNAi), has recently been described in a number of species including Drosophila. To ascertain if dsRNA-triggered RNAi is present in mosquito cells, we used Aedes albopictus C6/36 cells, and to investigate the feasibility of blocking viral gene expression and replication, we used two mosquito-borne viruses, Semliki Forest virus (SFV) and the serotype 1 dengue virus (DEN1). We demonstrate that dsRNA can specifically inhibit transgene expression in C6/36 cells from both plasmid and SFV replicons and can significantly modify the kinetics of DEN1 RNA and virus replication. The inhibition mediated by dsRNA was sequence-specific and either equal or superior to that induced by antisense single-stranded RNA (ssRNA). This study demonstrates dsRNA-triggered inhibition of gene expression and virus replication in mosquito cells and suggests that this mechanism could be used to block pathogen replication within an insect host and, thus, block disease transmission.