Aberrant acetylation has been strongly linked to tumorigenesis, and the modulation of acetylation through targeting histone deacetylases (HDACs) is gathering increasing pace as a viable therapeutic strategy. A genome-wide loss-of-function screen identified HR23B, which shuttles ubiquitinated cargo proteins to the proteasome, as a sensitivity determinant for HDAC inhibitor-induced apoptosis. HR23B also governs tumor cell sensitivity to drugs that act directly on the proteasome. The level of HR23B influences the response of tumor cells to HDAC inhibitors, and HR23B is found at high levels in cutaneous T cell lymphoma in situ, a malignancy that responds favorably to HDAC inhibitor-based therapy. These results suggest that deregulated proteasome activity contributes to the anticancer activity of HDAC inhibitors.

Autophagy plays a critical role in cancer formation and therapeutic resistance. However, little is known about how autophagy is regulated in cancer and how it mediates therapeutic resistance. Here we elect to use chronic myeloid leukemia (CML) as a cancer model to study autophagy in that it is driven by a single onco-protein BCR-ABL, whose activity can be selectively blocked by imatinib a front-line treatment for CML. Moreover, imatinib resistance frequently occurs in CML patients. Thus, unraveling autophagy regulation in CML and its role in overcoming imatinib resistance has substantial therapeutic benefits not only for CML but also for other cancers that can be treated by imatinib. In this report, we performed a genome-wide RNA interference screen in K562 human CML cells using monodansylcadaverine (MDC) that marks autolysosomes followed by fluorescence-activated cell sorting to label and isolate autophagic cells. We have identified 336 candidate genes, knockdown of which significantly ...

To elucidate the molecular mechanisms underlying pathogen-associated molecular pattern (PAMP)-induced defense responses in potato (Solanum tuberosum), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep-13, a PAMP from Phytophthora, induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive-like cell death. We have previously shown that SA is required for Pep-13-induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12- oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F-box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep-13 treatment, neither did they contain significant amounts of JA after elicitation. In response ...

Identification of components of the intracellular transport machinery of acylated proteins by a genome-wide RNAi screen [Elektronische Ressource] / presented by Julia Ritzerfeld : IDENTIFICATIO N O F CO M PO NENTS O F TH E INTRACELLU LAR TRANSPO RT M ACH INERY O F ACYLATED PRO TEINS BY A GENO M E‐W IDE RNAI SCREEN DISSERTATIO N submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto Caro la University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Julia Ritzerfeld

TY - CHAP. T1 - Short hairpin RNA-mediated gene silencing. AU - Lambeth, Luke S. AU - Smith, Craig A.. PY - 2013. Y1 - 2013. N2 - Since thefirst application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of shRNA precursors that are effectively processed by the RNAi pathway can lead to potent gene knockdown. The past decade has seen continual advancement and improvement to the various strategies that can be used for shRNA delivery, and the use of shRNAs for clinical applications is well underway. Driving these developments has been the many benefits afforded by shRNA technologies, including the stable integration of expression constructs for long-term expression, infection of difficultto-target cell lines and tissues using viral vectors, and the temporal control of shRNA transcription by inducible promoters. The use of different ...

RNA interference (RNAi) is a powerful tool to study the intracellular membrane transport and membrane organelle behavior in the nematode Caenorhabditis elegans. This model organism has gained popularity in the trafficking field because of its relative simplicity, yet being multicellular. C. elegans is fully sequenced and has an annotated genome, it is easy to maintain, and a growing number of transgenic strains bearing markers for different membrane compartments are available. C. elegans is particularly well suited for protein downregulation by RNAi because of the simple but efficient methods of dsRNA delivery. The phenomenon of systemic RNAi in the worm further facilitates this approach. In this chapter we describe methods and applications of RNAi in the field of membrane traffic. We summarize the fluorescent markers used as a readout for the effects of gene knockdown in different cells and tissues and give details for data acquisition and analysis ...

RNA interference (RNAi) is a gene-silencing mechanism by which a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) and a double-stranded (ds) short-interfering RNA (siRNA), targets a complementary mRNA for site-specific cleavage and subsequent degradation. While longer dsRNA are endogenously processed into 21- to 24-nucleotide (nt) siRNAs or miRNAs to induce gene silencing, RNAi studies in human cells typically use synthetic 19- to 20-nt siRNA duplexes with 2-nt overhangs at the 3-end of both strands. Here, we report that systematic synthesis and analysis of siRNAs with deletions at the passenger and/or guide strand revealed a short RNAi trigger, 16-nt siRNA, which induces potent RNAi in human cells. Our results indicate that the minimal requirement for dsRNA to trigger RNAi is an approximately 42 A A-form helix with approximately 1.5 helical turns. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene,

DasGupta et al. [3] developed a high-throughput assay based on the known ability of canonical Wnt signaling to activate transcription of luciferase reporter constructs in transfected cells. Improving on the widely used construct TOP-Flash [13], they generated two new reporters each containing multiple TCF-binding sites upstream of a different minimal promoter. Because only the TCF sites were common between the reporters, off-target effects unrelated to β-catenin/TCF signaling were minimized. Reporters with mutated TCF-binding sites also served as specificity controls. The authors first validated the behavior of these reporters in transfection assays of Drosophila cell lines. Then they scaled up the transfections to incorporate approximately 22,000 double-stranded RNAs (dsRNAs), so as to induce RNAi [3], and tested the individual effects on Wingless-induced signaling. The library of dsRNA sequences, previously used in other high-throughput RNAi screens, is directed at all known open reading ...

RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cells cytoplasm, where they interact with the catalytic RISC component argonaute.[5] When the dsRNA is exogenous (coming from infection by a virus with an RNA genome or laboratory manipulations), the RNA is imported directly into the cytoplasm and cleaved to short fragments by Dicer. The initiating dsRNA can also be endogenous (originating in the cell), as in pre-microRNAs expressed from RNA-coding genes in the genome. The primary transcripts from such genes are first processed to form the characteristic stem-loop structure of pre-miRNA in the nucleus, then exported to the cytoplasm. Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC.[6] Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer,[7] which binds and cleaves double-stranded RNAs (dsRNAs) in plants, or short hairpin ...

RNA interference (RNAi) is a naturally occurring phenomenon that results in the suppression of a target RNA sequence utilizing a variety of possible methods and pathways. To dissect the factors that result in effective siRNA sequences a regression kernel Support Vector Machine (SVM) approach was used to quantitatively model RNA interference activities. Eight overall feature mapping methods were compared in their abilities to build SVM regression models that predict published siRNA activities. The primary factors in predictive SVM models are position specific nucleotide compositions. The secondary factors are position independent sequence motifs (N-grams) and guide strand to passenger strand sequence thermodynamics. Finally, the factors that are least contributory but are still predictive of efficacy are measures of intramolecular guide strand secondary structure and target strand secondary structure. Of these, the site of the 5 most base of the guide strand is the most informative. The capacity of

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate ...

Researchers at the Medical University of South Carolina show in a new report that the RNA interference machinery, normally thought to reside in the nucleus or cytoplasm, predominantly localizes to these apical junctions and influences cell biology in the colon.

RNA interference has revolutionized our ability to study the effects of altering the expression of single genes in mammalian (and other) cells through targeted knockdown of gene expression. In this report we describe a web-based computational tool, siRNA Information Resource (sIR), which consists of a new open source database that contains validation information about published siRNA sequences and also provides a user-friendly interface to design and analyze siRNA sequences against a chosen target sequence. The siRNA design tool described in this paper employs empirically determined rules derived from a meta-analysis of the published data; it uses a weighted scoring system that determines the optimal sequence within a target mRNA and thus aids in the rational selection of siRNA sequences. This scoring system shows a non-linear correlation with the knockdown efficiency of siRNAs. sIR provides a fast, customized BLAST output for all selected siRNA sequences against a variety of databases so that the user

Ryan uses primary cell models (donated healthy live cells) and tissue samples from patients to investigate the cellular genetic workings of blood disorders and cancer. He tends to collect the RNA produced when genes are switched on and off, using cutting edge techniques to collect the sequences of these genes or uses microarray technologies to profile them. From this information he can identify key genes in a disease and use RNA interference technologies to switch variations of these genes off, inhibiting the production of controlling proteins for potential treatments in that disease.. As these RNA and protein molecules change in diseases we can use these changes to diagnose and help in clinical prognosis. Ryan has worked on novel sensor technologies to make such systems clinically acceptable, quicker and more sensitive.. Examples of disease work:. Manipulating gene switching in Sickle Cell Anaemia as a potential treatment; Dr D Carter, NCRNA and Chromatin Research Group, Oxford Brookes ...

Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients AML cells. Importantly, WT1-antigen T cell receptor(+) PD-1(+) 2D3 cells showed increased activation toward PD-L1 silenced WT1(+) AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-γ production of minor histocompatibility antigen-specific CD8(+) T ...

Previous works in the budding yeast S. cerevisiae and the fission yeast S. pombe have revealed that aslncRNAs are globally low abundant as they are extensively degraded by RNA surveillance machineries. For instance, the nuclear exosome targets a class of lncRNAs referred to as CUTs (Wyers et al, 2005; Neil et al, 2009; Xu et al, 2009), whereas the cytoplasmic 5′-3′ exoribonuclease Xrn1 degrades the so-called XUTs (Van Dijk et al, 2011), both types of transcripts being mainly antisense to protein-coding genes. However, this classification into CUTs and XUTs is not exclusive, some aslncRNAs being cooperatively targeted by the two RNA decay pathways. In fission yeast, an additional class of aslncRNAs (DUTs) was recently identified. DUTs accumulate in the absence of the ribonuclease III Dicer (Atkinson et al, 2018), highlighting the role of Dicer and RNAi in the control of aslncRNAs expression in fission yeast. This class of transcripts is absent in S. cerevisiae, which has lost the RNAi system ...

In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana ...

As the portfolio of RNAi methods continues to expand, options become available for even the most complex systems being studied. Until recently, synthetic siRNA was the RNAi vehicle most broadly applicable to a wide variety of systems and applications. With commercial suppliers designing and producing synthetic siRNAs, little manipulation is required for the consumer. This format is amenable to any scale of research being performed provided the system is easily transfected (e.g., standard transformed cell lines). However, obstacles for using synthetic siRNAs include being a non-renewable resource, the transient nature of silencing, and the difficulty faced in transfecting primary cells and non-dividing cell lines such as neurons, lymphocytes, and macrophages. In addition, in vivo knockdown studies are particularly cumbersome.. For those facing the above hurdles, DNA vector-based shRNA methods provide the necessary solutions. shRNA expression vectors may be propagated in Escherichia coli and, ...

Control of metabolic flux, the flow of metabolites through a complex metabolic network, is of importance to understand how an organism is sensing, and responding to, nutrient changes in its environment. Metabolic flux control can be measured for, and a control coefficient assigned to, each enzyme in a pathway. Measuring metabolic flux control in multicellular organisms is complicated by the fact that nutrient sensing and metabolic flux control may vary by tissue type. Major effects should be detectable in genomic information, as enzymes with high control coefficients will exhibit genetic patterns of adaptation when the pathway is under selection pressure. I used genetic variation within and among populations of Drosophila melanogaster, as well as divergence between D. melanogaster and the closely related D. simulans, to identify candidate genes for experimental study. I then conducted experiments with candidate genes using tissue specific RNA interference knockdown, focusing on two enzymes ...

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular ...

Bahiagrass is one of the most important warm season forage grasses. In Florida alone it is grown on more than 5 million acres. Howeve r, the high lignin content in the bahiagrass biomass significantly reduces its forage qual i ty. A key enzyme in the lignin biosynthetic pathway is th e 4coumarate CoA ligase (4CL); it catalyzes the formation of CoA thiol esters of 4 coumarate and other hydr oxycinn amates. We cloned four 4CL cDNA s from tetraploi d ba hiagrass cv. Argentine and an RNAi construct targeting a highly conserved domain was constructed using 200 bp of the coding sequences. The 4CL RNAi construct was intr o duced to bahiagrass callus by b iolistic gene transfer under transcriptional control of three alternative promoters: the constitutive e35S promoter, OsC4H promoter for xylem specific expression and the ZmdJ1 promoter for expre s sion in the green tissue. Following regeneration of plants their transgenic nature was confirmed using PCR and Southern blot analysis. Significant reduction ...

Many species, across a wide phylogenetic range, respond to aberrant/foreign RNA by degrading endogenous mRNA in a sequence-specific manner (1). This phenomenon, broadly referred to as posttranscriptional gene silencing (PTGS), can be triggered by the introduction of double-stranded RNA (dsRNA) [RNA interference (RNAi)], transformation with sense transgenes (cosuppression/quelling), or viral infection (2). RNAi acts as a cellular defense against parasitic nucleic acids and provides a fortuitous technique for biologists to reduce or eliminate a gene activity (3). RNAi-like mechanisms are also involved in the production of small noncoding RNAs that control developmental timing (4, 5). A better understanding of RNAi may then shed light on genome defense and endogenous developmental pathways.. The molecular mechanisms underlying RNAi are beginning to be elucidated. dsRNA is processed into small double-stranded fragments of 21-25 nucleotides, called small interfering RNA (siRNA; refs. 6-8), by the ...

Plants and fungi can use conserved RNA interference machinery to regulate each others gene expression-and scientists think they can make use of this phenomenon to create a new generation of pesticides.. 6 Comments. ...

Supplemental Figure 2 - Fig. S2. Quantification of effects on cells after CAP-D2 RNAi. (A) Growth curve showing the number of cells at different time points after CAP-D2 dsRNA treatment. Cells grew more slowly, plateaued at 72 hours, and did not change significantly after that time. (B) The percentage of mitotic cells in control and CAP-D2 RNAi cells. The percentage of mitotic cells increased two- to threefold in the CAP-D2 RNAi between 36 and 72 hours (6.7% versus 2.2% at 48 hours). (C) The percentage of abnormal mitotic cells in control and CAP-D2 RNAi cells. The majority of mitotic cells are abnormal 36 hours and later after dsRNAi treatment. (D) Histogram showing the percentage of cells in prometaphase after staining for Cyclin B/P-H3/a-tubulin in control and CAP-D2 RNAi cells. Cells delay in prometaphase in the CAP-D2-depleted cells. (E) Histogram showing the percentage of cells in anaphase after staining for Cyclin B/P-H3/a-tubulin, in control and CAP-D2 RNAi cells. The anaphase index in ...

https://doi.org/10.18632/oncotarget.4817 Aleksandra A. Pandyra, Peter J. Mullen, Carolyn A. Goard, Elke Ericson, Piyush Sharma, Manpreet Kalkat, Rosemary Yu, Janice T. Pong, Kevin R. Brown, Traver...

The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of ...

The pSUPER.retro (Oligoengine) RNA interference system was used to achieve stable expression of siRNAs. Oligonucleotides targeted to calpain 2 or PTP1B mRNA as well as a nonsilencing control were synthesized by Integrated DNA Technologies, annealed, and cloned into the pSUPER.retro.puro vector according to manufacturers instructions. Retroviral transfection was performed as described previously (Franco et al., 2004a). Wild-type MTLn3 cells were infected at 32°C for 6 h and allowed to recover in growth medium for 24 h before selection with 1 μg/ml puromycin for 4-5 d. Target sequences for calpain 2 in MTLn3 cells: control, 5′-TTCTCCGAACGTGTCACGT-3′; Capn2 si-A, 5′-AGGCCTATGCCAAGATCAA-3′; and Capn2 si-B, 5′-GAATGGCGATTTCTGCATC-3′. Target sequences for PTP1B in MTLn3 cells: PTP1B si-A, 5′-GCTGACACTGATCTCTGAA-3′; and PTP1Bsi-B, 5′-CAGGAGGAGCCTTGGTGTC-3′. Target sequences for human calpain 2 have been described previously (Su et al., 2006). Target sequences for cortactin: ...

Background: While genetic knockdown of RAS in mouse tumor models has substantiated it as a therapeutic target, there is no effective means of targeting RAS currently available in the clinic today. Numerous RNA interference-based studies targeting RAS have demonstrated therapeutic effects, however, effective delivery has been a major obstacle that has impeded this approach.. U1 Adaptors are a novel technology for oligonucleotide-mediated gene silencing that act by selectively interfering with polyadenylation of messenger RNA (mRNA) inside the cell nucleus. Polyadenosine (PolyA) tail addition is an obligatory step in mRNA maturation and function, and its failure results in rapid degradation of the nascent message by endogenous nucleases. The eukaryotic U1 small nuclear ribonucleoprotein complex (U1 snRNP) is best known for its role as a pre-mRNA splicing factor, but also acts naturally to silence some genes by suppressing polyadenylation.. U1 Adaptors are synthetic oligonucleotides that enable the ...

Ola R, Dubrac A, Han J, Zhang F, Fang JS, Larrivée B, Lee M, Urarte AA, Kraehling JR, Genet G, Hirschi KK, Sessa WC, Canals FV, Graupera M, Yan M, Young LH, Oh PS, Eichmann A: PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia. Nat Commun. 2016 Nov 29; 2016 Nov 29. PMID: 27897192 Zhang F, Prahst C, Mathivet T, Pibouin-Fragner L, Zhang J, Genet G, Tong R, Dubrac A, Eichmann A: The Robo4 cytoplasmic domain is dispensable for vascular permeability and neovascularization. Nat Commun. 2016 Nov 24; 2016 Nov 24. PMID: 27882935 Kraehling JR, Chidlow JH, Rajagopal C, Sugiyama MG, Fowler JW, Lee MY, Zhang X, Ramírez CM, Park EJ, Tao B, Chen K, Kuruvilla L, Larriveé B, Folta-Stogniew E, Ola R, Rotllan N, Zhou W, Nagle MW, Herz J, Williams KJ, Eichmann A, Lee WL, Fernández-Hernando C, Sessa WC: Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells. Nat Commun. 2016 Nov 21; 2016 Nov 21. PMID: 27869117 ...

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In this paper, we describe the results of a knockdown screen in mouse ES cells to identify factors required for differentiation. Grouping of the identified genes into functional pathways shows that multiple hits are involved in Ras-Mek-Erk signaling. EphB4 receptors can regulate the activity of the Ras family of GTPases, including H-Ras and R-Ras (Zou et al., 1999; Miao et al., 2001; Wang et al., 2006). When Ptpn11 (also called Shp-2), another hit from our screen, was prevented from interacting with a mutated gp130 receptor that failed to activate ERKs, this led to self-renewal (Burdon et al., 1999). These data show that our unbiased, genome-wide knockdown approach identified several factors that were previously identified to be important in self-renewal of ES cells and validate our screening strategy. An shRNA against Capn10 was found in ∼50% of the sequences and, when tested individually, this shRNA showed strong ES colony outgrowth during the first 2 wk after removal of LIF. During the 3rd ...

TY - JOUR. T1 - RNAi as a potential new therapy for HIV infection. AU - Wheeler, Lee A.. AU - Dykxhoorn, Derek M.. PY - 2008/12/1. Y1 - 2008/12/1. N2 - Controlling HIV infection continues to be a major clinical and scientific challenge. Despite the therapeutic benefits associated with HAART, the need for novel treatment approaches to combat HIV-1 remains. Effective inhibition of HIV-1 infection has been achieved by harnessing the endogenous RNAi pathway in a variety of cell types, including primary T cells and macrophages. Here we discuss the opportunities and challenges associated with translating these findings into clinically relevant therapeutic approaches.. AB - Controlling HIV infection continues to be a major clinical and scientific challenge. Despite the therapeutic benefits associated with HAART, the need for novel treatment approaches to combat HIV-1 remains. Effective inhibition of HIV-1 infection has been achieved by harnessing the endogenous RNAi pathway in a variety of cell types, ...

The field of RNA-based gene regulation has been attracting increasing interest over the past couple of years, and the regulation of gene expression by small dsRNAs is being studied intensively. Such interference can be mediated by siRNAs, which cleave a sequence-specific target mRNA, or by micro-RNAs, which inhibit translation of a target mRNA. Noncoding RNAs have also been found to play important roles in the regulation of gene expression, for example, in gene silencing by methylation of DNA or histones. Small interfering RNAs are expected to have medical application in human therapy as drugs with high specificity for their molecular targets.. A number of studies on synthetic siRNAs or DNA vector-derived small hairpin RNAs (shRNAs) in cell culture systems have been published, and there are also several animal studies (15, 16, 17, 18, 19) . McCaffrey et al. (15) cotransfected the firefly luciferase gene along with synthetic siRNAs or a shRNA expression vector into mice by hydrodynamic injection ...

RNAi is a convenient, widely used tool for screening for genes of interest. We have recently used this technology to screen roughly 750 candidate genes, in C. elegans, for potential roles in regulating muscle protein degradation in vivo. To maximize confidence and assess reproducibility, we have only used previously validated RNAi constructs and have included time courses and replicates. To maximize mechanistic understanding, we have examined multiple sub-cellular phenotypes in multiple compartments in muscle. We have also tested knockdowns of putative regulators of degradation in the context of mutations or drugs that were previously shown to inhibit protein degradation by diverse mechanisms. Here we discuss how assaying multiple phenotypes, multiplexing RNAi screens with use of mutations and drugs, and use of bioinformatics can provide more data on rates of potential false positives and negatives as well as more mechanistic insight than simple RNAi screening.

RNAi is a convenient, widely used tool for screening for genes of interest. We have recently used this technology to screen roughly 750 candidate genes, in C. elegans, for potential roles in regulating muscle protein degradation in vivo. To maximize confidence and assess reproducibility, we have only used previously validated RNAi constructs and have included time courses and replicates. To maximize mechanistic understanding, we have examined multiple sub-cellular phenotypes in multiple compartments in muscle. We have also tested knockdowns of putative regulators of degradation in the context of mutations or drugs that were previously shown to inhibit protein degradation by diverse mechanisms. Here we discuss how assaying multiple phenotypes, multiplexing RNAi screens with use of mutations and drugs, and use of bioinformatics can provide more data on rates of potential false positives and negatives as well as more mechanistic insight than simple RNAi screening.

The yeast Efr3p protein is a major regulator of the Stt4p phosphatidylinositol 4-kinase at ER-PM contact sites. Its mutant fly homologue, Rbo displays diminishing light responses attributed to progressively impaired PLC signaling. Here we find that Efr3s play a role in maintaining responsiveness to angiotensin II (AngII) receptors. RNAi-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca2+ response to high concentration of AngII in HEK293 cells expressing the wild type but not a truncated AT1a receptor, missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. Similar but smaller effect of EFR3 depletion was ...

Both ATM and ATR display a preference for phosphorylating SQ/TQ motifs in their substrates (Kim et al., 1999; Traven and Heierhorst, 2005; Shiloh, 2006). ATR is predominantly activated by UV light and stalled replication forks, whereas ATM is specifically activated by DSBs of DNA, as seen after irradiation, etoposide, or oxidative stress (Abraham, 2001; Shiloh, 2006). In contrast, treatment with the ATP-competitive kinase inhibitor, staurosporine, does not activate ATM or affect the phosphorylation status of ATM-dependent substrates (Kamer et al., 2005). We show here that DNA-damaging agents, such as IR and etoposide, trigger MEF2D phosphorylation. Moreover, MEF2D phosphorylation only increased after etoposide exposure in wt-ATM cells but not in ATM-deficient cells. These results suggest that ATM mediates MEF2D phosphorylation in response to DSBs in DNA.. Furthermore, in the present study, RNA interference-mediated knockdown experiments in cerebellar granule cells indicate that endogenous MEF2D ...

RNA interference (RNAi) is an important pathway that is used in many different organisms to regulate gene expression. This animation introduces the principles of RNAi involving small interfering RNAs (siRNAs) and microRNAs (miRNAs). We take you on an audio-visual journey through the steps of gene expression and show you an up-to-date view of how RNAi can silence specific mRNAs in the cytoplasm.. ...

Qiang Zhang is the author of this article in the Journal of Visualized Experiments: DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference involves the targeted knockdown of mRNA triggered by complementary dsRNA molecules applied to an experimental organism. Although this technique has been successfully used in honeybees

RNA interference (RNAi) therapeutics (siRNA, miRNA, etc.) represent an emerging medicinal remedy for a variety of ailments. However, their low serum stability and low cellular uptake signi cantly restrict their clinical applications. Exosomes are biologically derived nanodimensional vesicle ranging from a few nanometers to a hundred. In the last few years, several reports have been published demonstrating the emerging applications of these exogenous membrane vesicles, particularly in carrying different RNAi ther- apeutics to adjacent or distant targeted cells. In this report, we explored the numerous aspects of exosomes from structure to clinical implications with special emphasis on their application in delivering RNAi-based therapeutics. siRNA and miRNA have attracted great interest in recent years due to their speci c applica- tion in treating many complex diseases including cancer. We highlight strategies to obviate the challenges of their low bioavailability for gene therapy ...

The implementation of decisions affecting cell viability and proliferation is dependant on prompt detection of the problem to become addressed, formulation and transmission of the correct group of instructions and fidelity in the execution of orders. nearing mitosis might encounter, presenting the effect of post-translational adjustments (PTMs) on the right and timely working of pathways fixing errors or harm before chromosome segregation. We conclude this article having a perspective on the existing position of mitotic signaling pathway inhibitors 154235-83-3 IC50 and their potential make use of in malignancy therapy. (Mazzarello, 1999). The main occasions characterizing changeover through the cell routine are cell development, where means cells boost their size and the amount of organelles, and duplication of hereditary materials in S-phase. If not really perturbed, upon conclusion of DNA replication cells enter mitosis, a term that originally explained nuclear department (Mazzarello, 1999). ...

The most enjoyable part in following RNAi Therapeutics is to look at the rich stream of scientific data and determine the absolute maturity and competitive position of the technologies and companies involved, as well as getting a glimpse at relationship dynamics. I therefore thought to share today two examples of this that I picked up recently. One is a paper by Sirna Therapeutics/Merck shedding some light on their approach towards RNAi pharmacology and RNAi trigger design. The other is some intriguing evidence that Silence Therapeutics most important gene target, PKN3, is gaining traction in the pharmaceutical space. Studying the pharmacology of siRNA delivery. Pei and colleagues from Merck published in RNA a nice paper on better understanding the pharmacology of siRNA delivery [Pei et al. (2010). Quantitative evaluation of siRNA delivery in vivo]. Unlike small molecules or even antibodies, the pharmacology of RNAi Therapeutics is more complex as simply measuring the raw tissue abundance of an ...

Keywords: placenta, cancer of the colon, endothelium, VEGF, immunohistochemistry, angiogenesis Vascular endothelial development element A (VEFG-A), probably the most prominent person in the VEGF family, is one of the key regulators of angiogenesis in general, including the promotion of tumor progression and metastasis (Kim et al. 1993; Ferrara et al. 2003). The important role of this growth factor in different areas of biological sciences makes it therefore an interesting target in many immunohistochemical studies. At present, at least nine different primary anti-VEGF antibodies are commercially available that can be applied to formalin-fixed and paraffin-embedded tissue samples (Table 1). Considering the literature on VEGF IHC applications, there is surprisingly little discussion about the selection of the applied VEGF antibody, and no consensus on which VEGF antibody is most reliable. In an attempt to validate five VEGF antibodies, Zhang et al. (1998) reported the R and D Systems mouse ...

Caenorhabditis elegans have many benefits for genetic manipulation and research. One of the most beneficial features is that it is transparent. This is great for microscopy because it makes it easier for us to see what is different with the worms reproductive system when comparing it to the normal, not treated worm. For the experiments I perform for the microscopy element, we repeat the RNAi interference experiments with strains with fluorescent markers. GFPs are green fluorescent proteins that can stain a particular part of a cell; like a cell wall and RFP are red fluorescent protein can stain the chromosomes within the nucleus of the cell. With the strain I am working with, AJ740, I can utilize the GFP and RFP to see what is happening to the shape and overall placement of the eggs within the affect mother worm treated through RNA interference along with what is going on with the chromosomes. I have several questions. General questions like: are the eggs going to the right place and are there ...

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TOPICS-I. 1-Regulatory RNA, 2- RNA interference and micro RNA, 3-Retroviruses, 4-Transposons and Retroposons, 5-Promoters and Enhancers , 6-Activating Transcription, 7-RNA Splicing and Processing, 8-Chromosomes-Nucleosomes, 9-Controlling Chromatin Remodeling and Structure. Slideshow 6603499 by angelica-figueroa

One siRNA sequence, many cell lines - posted in siRNA, microRNA and RNAi: Hi all, Im new to process of siRNA transfection and I was wondering: Will one siRNA sequence (previously validated in the lab) be good enough to transfect multiple other cell lines from the same organism? I understand that the actual process of transfection will be different for each cell line, but I am curious as to whether I need to also worry about the sequence itself. Thanks!

Press release - Allied Market Research - RNA Interference (RNAi) Drug Delivery Market Statistics (2019-2026): Hyper Growth Recorded in the Future - published on openPR.com

Furfural is a major growth inhibitor in lignocellulosic hydrolysates and improving furfural tolerance of microorganisms is critical for rapid and efficient fermentation of lignocellulosic biomass. In this study, we used the RNAi-Assisted Genome Evolution (RAGE) method to select for furfural resistant mutants of Saccharomyces cerevisiae, and identified a new determinant of furfural tolerance. By using a genome-wide RNAi (RNA-interference) screen in S. cerevisiae for genes involved in furfural tolerance, we identified SIZ1, a gene encoding an E3 SUMO-protein ligase. Disruption of SIZ1 gene function by knockdown or deletion conferred significantly higher furfural tolerance compared to other previously reported metabolic engineering strategies in S. cerevisiae. This improved furfural tolerance of siz1Δ cells is accompanied by rapid furfural reduction to furfuryl alcohol and leads to higher ethanol productivity in the presence of furfural. In addition, the siz1Δ mutant also exhibited tolerance towards

In metazoans, the hematopoietic system plays a key role both in normal development and in defense of the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations leading to melanotic mass (or tumor) formation in larvae have been associated to the deregulated differentiation of lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and UAS-dsRNA transgenic lines. By targeting around 10% of the Drosophila genes, this in vivo RNA interference screen allowed us to recover 59 melanotic tumor suppressor genes. In line with previous studies, we

Root lesion nematodes (RLNs, Pratylenchus spp.) are economically important migratory endoparasitic pests of crop plants. The overall aim of the work in this thesis was to undertake molecular studies on Pratylenchus thornei to characterise genes potentially involved in plant parasitism, and to determine if they are amenable to gene silencing (RNA interference). The recent availability of the transcriptomes of three RLN species, Pratylenchus coffeae, P. thornei and P. zeae significantly expanded the resources available for this study. Amongst the transcriptome data, putative parasitism genes (PPGs) were identified. A common assembly platform was also used to analyse transcriptome data to determine whether differences between PPGs identified here and those reported previously were consistent when the same assembly was used. Bioinformatic analysis was also undertaken to compare PPGs between different Pratylenchus spp. The results showed that there were some differences in the PPGs identified in P. ...

RNA interference (RNAi) is a powerful technology used to knock down genes in basic research and medicine. In 2006 RNAi technology using Caenorhabditis elegans (C. elegans) was awarded the Nobel Prize in medicine and thus students graduating in the biological sciences should have experience with this technology. However, students struggle conceptually with the molecular biology behind the RNAi technology and find the technology difficult to grasp. To this end, we have provided a simple, streamlined and inexpensive RNAi procedure using C. elegans that can be adopted in upper level biology classes. By using an unknown RNAi-producing bacteria, students perform novel techniques, observe and determine which mystery gene was knocked down based on phenotype and experience a new research organism. By bringing this technology to the undergraduate lab bench, the gap between blackboard concept and proof of concept can be bridged.

The second table includes only studies involving plant-mediated RNAi. Seven of the nine studies referenced involved the use of Myzus persicae feeding on transgenic tobacco or Arabidopsis and targeting a variety of genes. All of those studies reported some negative impact on the fecundity of insects feeding on these plants.. Finally, the authors address challenges facing the successful implementation and adoption of plant-mediated RNAi for aphid control. A notable challenge is the presence of dsRNAases in the gut of at least the pea aphid. Furthermore, the authors note that interactions between the aphids RNA interference machinery and the plant viruses they transmit could impact the RNAi response to plant-mediated RNAi and this is one of a number of areas that needs further study.. Producing dsRNA and delivering it to the phloem is a challenge and will benefit from the use of phloem-specific promoters and enhancers, according to the authors. Of course, the potential for off-target effects and ...

Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers virulent sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism. ...

article{75a89636-8dd7-4602-9e06-cb1161b401a9, abstract = {Tissue factor (TF) has been implicated in the thrombotic complications seen during vascular rejection of allografts and may contribute to intimal hyperplasia in chronic allograft vasculopathy. Downregulation of endothelial TF expression post-transplantation could therefore be of therapeutic value. Lentivirus-mediated RNA interference was used in primary endothelial cells (EC) to investigate its effects on TF protein expression and functional activity. Lentivirus-mediated expression of a TF-specific short-interfering (si) RNA with green fluorescent protein as a reporter gene (siRNATF-GFP) resulted in a 42 +/- 3.9% reduction in EC surface-expressed TF as compared with cells expressing a scrambled siRNATF sequence (P=0.025). The TF content in EC lysates was reduced from 6.85 +/- 1.99 ng to 3.05 +/- 0.82 ng (P=0.006). Factor X (FX) activation was not impaired on the apical EC surface. The subendothelial matrix of ECs with low TF expression ...

The heterotopic ossification of muscles, tendons, and ligaments is a common problem faced by orthopaedic surgeons. Runx2/Cbfa1 plays an essential role during the osteoblast differentiation and is considered as a molecular switch in osteoblast biology. RNA interference technology is a powerful tool for silencing endogenous or exogenous genes in mammalian cells. In this study, we investigated the effect of Runx2/Cbfa1-specific siRNA on osteoblast differentiation and mineralization in osteoblastic cells, and then constructed adenovirus containing siRNA against Runx2/Cbfa1 (Ad-Runx2-siRNA) to inhibit the formation of heterotopic ossification induced by BMP4, dernineralized bone matrix, and trauma in animal model. Our results showed that the Runx2/Cbfa1-specific siRNA could inhibit the expression of Runx2/Cbfa1 at the level of mRNA and protein. Analysis of the expression of osteoblast maturation genes including type I collagen, osteopontin, bone sialoprotein, and osteocalcin, alkaline phosphatase ...

BACKGROUND AND AIMS: Functional cure in chronic hepatitis B requires HBV DNA negativity, HBsAg loss and anti-HBs seroconversion. ARC-520 RNAi drug therapy targets cccDNA derived mRNA, including the full-length HBsAg transcript. Using a 19plex anti-HBs panel to map HBsAg epitopes, we have developed a predictive algorithm of an HBsAg clearance profile in patients undergoing HBsAg loss during tenofovir therapy, defined as reduced recognition at loop 1 and loop 2 HBsAg a determinant epitopes. Complimentary to this, we have developed assays to detect co-existing complexed anti-HBs (with HBsAg), and analysed the ARC- 520 cohorts with the aim of evaluating the impact of RNAi therapy on HBsAg loss, the identification of an HBsAg clearance profile and the development of co-existing anti-HBs ...

One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP ...

No effective chemotherapy for the treatment of malignant brain tumors, especially glioblastoma, exists so far. Despite the progress in surgical techniques and advances in the irradiation treatment, the concomitant chemotherapy is essential for the prevention of relapse and of major importance for patient outcome. The introduction of temozolomide combined with radiation in clinical practice led to slightly improved long-term survival, but malignant gliomas remain resistant to cancer chemotherapy. Thus, new strategies for the treatment of brain tumors are still needed. Objectives of this work were the evaluation of the TmHU protein as siRNA transfection reagent (Chapter 2), investigations on new kinesin Eg5 inhibitors, which specifically inhibit cell division during mitosis (Chapter 3), and the exploration of new tariquidar analogs as ABCB1 and ABCG2 inhibitors (Chapter 4). Due to the selective down-regulation of oncogene expression by small interfering RNA, siRNAs are considered as promising ...

In this study, we describe a new in vivo genetic assay to allow the identification of factors that functionally interact with Drosophila CHD1. We performed a focused candidate gene screen and identified several genes, including SWI2 family members, as potential CHD1 interaction partners. Given that this is an RNAi-based screen, we took several steps to reduce the possibility of nonspecific effects. First, we used both VALIUM1 and VALIUM20 lines in order to test as many different hairpin RNAs for a candidate gene as possible. However, many of our chosen lines resulted in lethality in both of the RNAi systems, while the use of VALIUM20-based RNAi lines to target several genes (Rtf1, ISWI, Pc, ash1, and trx) resulted in lethality, most likely because the shRNAs used in those lines lead to more effective knockdown of the target gene than the VALIUM1-based lines (Ni et al. 2011). Second, given that long double-stranded RNAs can produce off-target effects, we limited our analysis to lines that were ...

RNA interference (RNAi) is a widespread and widely exploited phenomenon which has potential as a strategy for both the treatment of disease and pest control. RNAi results in down‐regulation of a specific gene in response to the production of small interfering RNAs (siRNAs). RNAi is one of a family of processes mediated by small non‐coding RNAs [1], [2]. In Caenorhabditis elegans, and in a number of other organisms, RNAi is systemic so that the introduction of dsRNA into one tissue triggers gene silencing in other tissues [3], [4], [5], [6], [7]. Furthermore, systemic RNAi enables C. elegans and other organisms to exhibit environmental RNAi [5]. For example, feeding C. elegans on bacteria expressing dsRNA initiates a widespread RNAi response [8], [9]. Studies in C. elegans and other organisms have provided mechanistic insights into RNAi [4], [10], [11], [12], [13], although the role of exogenous RNAi in the normal life of C. elegans and other animals remains unclear [14].. Whilst C. elegans ...

TY - JOUR. T1 - Systemic delivery of siRNA by chimeric capsid protein. T2 - Tumor targeting and RNAi activity in vivo. AU - Choi, Kyung Mi. AU - Kim, Kwang Meyung. AU - Kwon, Ick Chan. AU - Kim, In-San. AU - Ahn, Hyung Jun. PY - 2013/1/7. Y1 - 2013/1/7. N2 - Recently, we reported that a chimeric capsid protein assembled into a macromolecular container-like structure with capsid shell and the resulting siRNA/capsid nanocarrier complexes efficiently suppressed RFP gene expression in the cell culture system. To extend RNAi to the in vivo applications, we here demonstrated that the siRNA/capsid nanocarrier complexes could have tumor-specific targeting ability in vivo as well as the increased stability of siRNA during body circulation. When systemically administered, our siRNA/capsid nanocarrier complexes delivered siRNA to tumor tissues and efficiently suppressed RFP gene expression in tumor-bearing mice. The enhanced longevity of siRNA in vivo could be explained by shielding effect derived from the ...

Spinocerebellar ataxia type 7 is a polyglutamine disorder caused by an expanded CAG repeat mutation that results in neurodegeneration. Since no treatment exists for this chronic disease, novel therapies such post-transcriptional RNA interference-based gene silencing are under investigation, in particular those that might enable constitutive and tissue-specific silencing, such as expressed hairpins. Given that this method of silencing can be abolished by the presence of nucleotide mismatches against the target RNA, we sought to identify expressed RNA hairpins selective for silencing the mutant ataxin-7 transcript using a linked SNP. By targeting both short and full-length tagged ataxin-7 sequences, we show that mutation-specific selectivity can be obtained with single nucleotide mismatches to the wild-type RNA target incorporated 3 to the centre of the active strand of short hairpin RNAs. The activity of the most effective short hairpin RNA incorporating the nucleotide mismatch at position 16 was

DNA-directed RNA interference (ddRNAi) is a gene-silencing technique that utilizes DNA constructs to activate an animal cells endogenous RNA interference (RNAi) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs (shRNA), that once processed bring about silencing of a target gene or genes. Any RNA, including endogenous mRNAs or viral RNAs, can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target. This mechanism has great potential as a novel therapeutic to silence disease-causing genes. Proof-of-concept has been demonstrated across a range of disease models, including viral diseases such as HIV, hepatitis B or hepatitis C, or diseases associated with altered expression of endogenous genes such as drug-resistant lung cancer, neuropathic pain, advanced cancer and retinitis pigmentosa. As seen in Figure 1, a ddRNAi construct encoding an shRNA is packaged into a delivery vector ...

Dicerna Pharmaceuticals, Inc., a biopharmaceutical company, focuses on the discovery and development of treatments for liver diseases and cancers based on a proprietary RNA interference technology platform in the United States and internationally. The companys development programs include DCR-PH1 for the treatment of primary hyperoxaluria type 1 (PH1) through targeting the gene encoding the liver enzyme glycolate oxidase; and other rare inherited diseases involving genes expressed in the liver. ...

The nematode worm Caenorhabditis elegans comprises an ancestral immune system. C. elegans recognizes and responds to viral, bacterial, and fungal infections. Components of the RNA interference machinery respond to viral infection, while highly conserved MAPK signaling pathways activate the innate im …

The 2006 Nobel Prize in Physiology or Medicine was awarded to Andrew Z. Fire and Craig C. Mello for their discovery of RNA interference (RNAi). RNAi is a biological process in which RNA molecules can silence (inhibit) or up- or down-regulate gene expression, typically by causing the destruction of specific messenger RNA (mRNA) molecules. This collection of articles begins with an introduction to RNAi, and proceeds to describe uses of this technology in various approaches to disease treatment, including gene therapy. Several laboratory protocols for silencing genes via RNAi are also provided, as are protocols for down-regulating and then rescuing those down-regulated genes, which demonstrates specificity of the approach.. This e-book - a curated collection from eLS, WIREs, and Current Protocols - offers a fantastic introduction to the field of RNA interference for students or interdisciplinary collaborators.. Table of Contents:. Introduction. Overview of Gene Silencing by RNA ...

RNA silencing processes are widespread in almost all eukaryotic organisms. They have various functions including genome protection, and the control of gene expression, development and heterochromatin formation. RNA interference (RNAi) is the post-transcriptional destruction of RNA, which is mediated by a ribonucleoprotein complex that contains, among several components, RNA helicases and Argonaute proteins. RNAi is functional in trypanosomes, protozoan parasites that separated very early from the main eukaryotic lineage and exhibit several intriguing features in terms of the control of gene expression. In this report, we investigated the functions of RNAi in Trypanosoma brucei. By searching through genome databases, novel Argonaute-like proteins were identified in several protozoa that belong to the kinetoplastid order, a group of organisms that diverged early from the main eukaryotic lineage. T. brucei possesses two Argonaute-like genes termed TbAGO1 and TbPWI1. Dual transient transfection assays

An important goal of contemporary HIV type 1 (HIV-1) research is to identify cellular cofactors required for viral replication. The HIV-1 Rev protein facilitates the cytoplasmic accumulation of the intron-containing viral gag-pol and env mRNAs and is required for viral replication. We have previously shown that a cellular protein, human Rev-interacting protein (hRIP), is an essential Rev cofactor that promotes the release of incompletely spliced HIV-1 RNAs from the perinuclear region. Here, we use complementary genetic approaches to ablate hRIP activity and analyze HIV-1 replication and viral RNA localization. We find that ablation of hRIP activity by a dominant-negative mutant or RNA interference inhibits virus production by mislocalizing Rev-directed RNAs to the nuclear periphery. We further show that depletion of endogenous hRIP by RNA interference results in the loss of viral replication in human cell lines and primary macrophages; virus production was restored to wild-type levels after

TY - JOUR. T1 - An effective gene-knockdown using multiple shRNA-expressing adenovirus vectors. AU - Motegi, Yukari. AU - Katayama, Kazufumi. AU - Sakurai, Fuminori. AU - Kato, Takuya. AU - Yamaguchi, Tomoko. AU - Matsui, Hayato. AU - Takahashi, Masahide. AU - Kawabata, Kenji. AU - Mizuguchi, Hiroyuki. PY - 2011/7/30. Y1 - 2011/7/30. N2 - Viral vectors expressing short hairpin RNA (shRNA) are attractive for efficient and tissue-specific RNA interference (RNAi) delivery. We and others previously reported that recombinant adenovirus (Ad) vector-mediated RNAi has great potential for a variety of applications in molecular biology studies and gene therapy. In the present study, we have developed an efficient Ad vector-mediated RNAi system, in which an Ad vector carries four shRNA-expression cassettes (Ad-multi-shRNA vector), a simple and effective strategy for enhancing the RNAi response per Ad vector particle. The data demonstrated that the Ad-multi-shRNA vectors showed an enhanced RNAi effect ...

Monsanto has been accused of silencing at least one scientist who explores the potential risks associated with the companys genetic modification technology called RNA interference (RNAi). Vicki Vance, Jr. Professor of Botany at the University of South Carolina, is a scientist who has contributed to the study of RNAi. RNAi lies at the heart of the debate on whether Monsanto is willing to admit the alleged hazards and the uncertainty associated with the technologies used to manufacture their products.. The point of this article is to reveal how Monsanto has attempted to suppress any credible scientific research containing human data that could lead to the further development on how human DNA reacts to the interference of genetically altered plant RNA through RNAi technology. The point is not to make any unsubstantiated claims that enough evidence exists to officially define this technology as dangerous to humans. The article should spark a discussion on the lack of understanding present in the ...

We are fortunate to perform translational biomedical research with dual goals: scientific discovery and the development of therapeutics for individuals with debilitating conditions. In providing a platform for gene modulation in the CNS, this publication had a large impact on our team and hopefully many others. Two of the first authors, Bruno Godinho and Matthew Hassler, are playing integral roles in building a new biotech company, Atalanta Therapeutics (https://www.atalantatx.com), focusing on the preclinical development of the di-siRNA. Julia Alterman is currently overseeing the development of a new direction at UMASS focusing on oligonucleotide therapeutics for multiple indications. I am working towards achieving my MD/PhD and returning to medical school in the spring. I hope to practice as a physician-scientist, aiding in the development and application of novel therapeutics for some of the most challenging diseases, specifically neurological conditions. The work in this publication truly ...

Metastasis is the leading cause of death for cancer patients. Consequently it is imperative that we improve our understanding of the molecular mechanisms that underlie progression of tumour growth towards malignancy. Advances in genome characterisation technologies have been very successful in identifying commonly mutated or misregulated genes in a variety of human cancers. A major challenge however is the translation of these findings to new biological insight due to the difficulty in evaluating whether these candidate genes drive tumour progression. Using the genetic amenability of Drosophila melanogaster we generated tumours with specific genotypes in the living animal and carried out a detailed systematic loss-of-function analysis to identify numerous conserved genes that enhance or suppress epithelial tumour progression. This enabled the discovery of functional cooperative regulators of invasion and the establishment of a network of conserved invasion suppressors. RNAi line: 12950Ra1 ...

Metastasis is the leading cause of death for cancer patients. Consequently it is imperative that we improve our understanding of the molecular mechanisms that underlie progression of tumour growth towards malignancy. Advances in genome characterisation technologies have been very successful in identifying commonly mutated or misregulated genes in a variety of human cancers. A major challenge however is the translation of these findings to new biological insight due to the difficulty in evaluating whether these candidate genes drive tumour progression. Using the genetic amenability of Drosophila melanogaster we generated tumours with specific genotypes in the living animal and carried out a detailed systematic loss-of-function analysis to identify numerous conserved genes that enhance or suppress epithelial tumour progression. This enabled the discovery of functional cooperative regulators of invasion and the establishment of a network of conserved invasion suppressors.. RNAi line: 37721 (III ...