BioAssay record AID 1224826 submitted by Iain Fraser: Genome-wide siRNA screen of genes regulating the Lipopolysaccharide-induced NF-kappaB and TNF-alpha responses in mouse macrophages_Primary screen TNF readout.
article{5993109, author = {De Backer, Lynn and Braeckmans, Kevin and Stuart, Marc CA and Demeester, Jo and De Smedt, Stefaan and Raemdonck, Koen}, issn = {0168-3659}, journal = {JOURNAL OF CONTROLLED RELEASE}, keyword = {DRUG-DELIVERY,EXOGENOUS SURFACTANT,LUNG,THERAPEUTICS,THERAPIES,SMALL-INTERFERING RNA,LOADED DEXTRAN NANOGELS,POLYMER HYBRID NANOPARTICLES,Targeted delivery,Pulmonary surfactant,siRNA delivery,Lung,Hybrid nanoparticles,Dextran nanogels,PLATFORM,CARRIERS}, language = {eng}, pages = {177--186}, title = {Bio-inspired pulmonary surfactant-modified nanogels: a promising siRNA delivery system}, url = {http://dx.doi.org/10.1016/j.jconrel.2015.03.015}, volume = {206}, year = {2015 ...
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,
Gene targets of RNAi reagents which are identified as hit in a RNAi screening and flagged as active in the corresponding PubChem BioAssay ...
DESCRIPTION (provided by applicant): The proposed research will enable mammalian cells to be used in mutation/selection experiments to identify genes involved in cellular processes. The objective of the proposal is to develop procedures for preparing siRNA libraries using genomic DNA and cDNA samples as the source material for siRNA templates. Libraries of siRNA vectors could comprise 1,000,000,000 to 1,000,000,000,000 unique siRNA expression vectors, making it possible to cover entire mammalian genomes with overlapping siRNAs. Mammalian cell populations transfected or transduced with siRNA vector libraries will be placed under selection to create cell populations expressing a desirable phenotype. The siRNA vector(s) integrated in the genomes of the selected cells will be amplified, cloned, and sequenced to reveal the siRNA template sequence. The siRNA sequences will be used to identify the genes whose down-regulation create the phenotype that was selected. The siRNA vector libraries should ...
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
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 ...
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 ...
The time course of lipophilic siRNA accumulation in KB-8-5 cells. The incubation time after carrier-free transfection of cholesterol-conjugated siMDR with hexyl
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, ...
Results Green fluorescence was observed in the cells transfected of negative control siRNA group though the fluorescence microscope. Compared with the blank control group, The MTT assay determined that the survival rate of H9C2 was decreased (p , 0.05) after the injured by hypoxia. And the results of flow cytometry showed that hypoxia increased cell apoptosis rate (P , 0.01) and the concentration of calcium (p , 0.01), while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P , 0.05 or P , 0.01). Compared with the hypoxia group, the transfection of Bim-siRNA increased the cell survival rate, decreased cell apoptosis rate and the concentration of calcium (p , 0.01 or p , 0.05). While there was no significant difference among Hypoxia Group, Hypoxia + Negative Control siRNA Group and Hypoxia + Mock control Group (p , 0.05). The results of Western blotting showed that the transfection of Bim-siRNA reduced the expression of Bim obviously (p , 0.01); meanwhile, reduced the ...
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 ...
Semple, SC, Akinc, A, Sandhu, A, Mui, B, Chow, C, Sah, D, Stebbing, D, Crosley, E, Hafez, I, Dorkin, JR, Qin, J, Lam, K, Wong, K, Nechev, L, Eisenhardt, ML, Jayaraman, M, Kazem, M, Maier, M, Srinivasulu, M, Weinstein, M, Chen, Q, Alvarez, R, Barros, S, Klimuk, SK, Borland, T, Kosovrasti, V, Tam, Y, MacLachlan, I, Manoharan, M, Ciufolini, MA, Tracy, M, de Fougerolles, A, Cullis, PR, Madden, TD, Hope, ...
mTOR and Rictor mediate cell migration.A: siRNA-induced mTOR depletion inhibits Tsc2−/− MEF migration. Tsc2−/− MEFs were transfected with siRNA mTOR or
Researchers working in the field of siRNA based therapeutics for viruses have generated a vast amount of data over the years. However, bioinformatics resources in the field were lacking and there was no viral siRNA efficacy prediction method available. In this direction, we have recently developed a comprehensive viral siRNA database VIRsiRNAdb [48] and another HIVsirDB [49] exclusively for HIV. Now we have developed VIRsiRNApred -a viral siRNA efficacy prediction algorithm.. Although many mammalian siRNA prediction algorithms have been developed in the past [33], these methods either classify a siRNA as effective/non-effective [29] or predict the inhibition efficacy of a siRNA [31, 32]. However, there is limited success in predicting siRNA efficacy due to limited size and diversity of available siRNA datasets [50].. Mammalian siRNA efficacy prediction methods were initially developed using siRNA tested under heterogeneous experimental conditions like Saetrom (581 siRNA), Shabalina (653 ...
GAITHERSBURG, Md., June 29 /PRNewswire/ -- Sirnaomics Receives Two SBIR Grants from NIH for Developing siRNA Therapeutics to Treat Glioblastoma and...
Suzhou Ribo Life Sciences is developing siRNA therapeutics for a range of diseases of high unmet need in China, including liver fibrosis, HIV, liver and
You can add the following controls to your FlexiPlate siRNA plate: AllStars Negative Control siRNA, AllStars Cell Death Control siRNA, Negative Control siRNA, Human GAPDH siRNA, Human Beta-Actin siRNA, Human and mouse MAPK1 siRNA, Human or mouse Lamin A/C siRNA, Mouse AKT1 siRNA, or other siRNAs from GeneGlobe, such as HP Validated siRNAs. ...
RNA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans-acting siRNAs are sh …
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!
Our study supports a previous conjecture that primary RdDM is required to initiate 24‐nt secondary siRNA formation. This requirement was initially suggested by the absence of secondary siRNAs in nrpe1 and drd1 mutants, which still produce primary siRNAs and the overlapping nascent RNA but lack primary RdDM (Kanno et al, 2008). The nascent RNA stably accumulates in the absence of primary RdDM in non‐silenced plants but, in that instance, is presumably a Pol II transcript (Figure 6) because it is still made in an nrpd1 mutant. The key function of primary RdDM appears to be in attracting the secondary siRNA‐generating machinery, which includes Pol IV and RDR2. The contribution may be direct if primary RdDM provides a template that is preferentially recognized by Pol IV, which has been proposed to transcribe methylated DNA (Herr et al, 2005; Onodera et al, 2005). In this model, Pol IV would transcribe the nascent RNA from the methylated template (Figure 6). An indirect contribution of primary ...
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 ...
The discovery of RNA interference (RNAi) in eukaryotic cells has opened new possibilities for drug development. Target-specific gene silencing via short interfering RNA (siRNA) displays many advantages over traditional pharmaceuticals. The unique siRNA nucleotide sequence benefits in higher drug specificity. Besides designing target-optimized siRNA oligonucleotides, one of the major challenges is to establish the efficient delivery system facilitating selective targeting altered tissues. The proper sequence-delivery combination provides these innovative therapeutics with unrivalled specificity and tolerability.. Another pivotal issue which has to be taken into account designing siRNA drugs is to provide them with an appropriate pharmacokinetic and pharmacodynamic properties. This is often achieved by an introduction of variety of chemical modifications into the siRNA molecules e.g. O-methyl groups added to the 2′ position of the ribosyl ring.. The ongoing extensive studies on RNAi resulted in ...
In recent years, small interference RNAs (siRNAs) have greatly enhanced our understanding of protein functions by allowing knockdown of targeted proteins at the mRNA level
After more than 20 years of research, we are now witnessing a breakthrough of small interfering RNA (siRNA)-based therapies. In 2018, the first-ever siRNA drug, Onpattro, reached the market, followed by the approval of Givlaari in 2019, and many other clinical trials are in progress. Holding the potential to treat a wide range of diseases from cancer to immunological disorders, siRNA therapeutics have received plenty of attention. With the support of a suitable delivery system, they can be directed to downregulate a specific target gene. Both approved siRNA drugs - Onpattro and Givlaari - are only able to reach the liver, however. Other organs that can be treated by loco-regional administration, such as the lung, are, in principle, good targets for siRNA therapies as well. In this view, siRNA-based drugs could not only act as an ally in the battle against the current COVID-19 pandemic but also against other severe lung diseases such as asthma. Despite the great advances in asthma treatment, this ...
P5 six rat DRG neurons were diluted to 35,000 cells ml in SATO media and handled with one mM dbcAMP, ten ug ml SLPI, or perhaps a volume of beads containing ten ug ml of SLPI protein. Cells were plated on CHO cell monolayers and incubated for 15 hours at 37 C. Neurons have been immunostained for BIII tubulin and neurite outgrowth was quantified as described above. siRNA experiments Accell SMARTpool rat Sunitinib price Smad2 siRNA, Accell Green Non Focusing on siRNA, and Accell Non Focusing on siRNA had been reconstituted to 100 uM working with one siRNA buffer. To assess transfection efficiency, P6 CGN and P1 rat cortical neurons have been prepared as described previously and diluted in supplemented Neurobasal A media. Neurons have been plated in poly L lysine coated 8 very well chamber slides at a density of 75,000 cells nicely. 24 hours later, the culture media in every properly was replaced with a hundred ul of 1 uM Accell Green Non Targeting siRNA in Accell siRNA delivery media. The neurons ...
Ribonucleic acid (RNA) is a ubiquitous family of large biological molecules that performs multiple vital roles in the coding, decoding, regulation, and expression of genes. Together with DNA, RNA comprises the nucleic acids, which, along with proteins, constitute the three major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but is usually single-stranded. Cellular organisms use messenger RNA (mRNA) to convey genetic information (recorded using the letters G, A, U, and C for the nucleotides guanine, adenine, uracil and cytosine) that directs synthesis of specific proteins, while many viruses encode their genetic information using an RNA genome.. Some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these active processes is protein synthesis, a universal function whereby mRNA molecules direct the assembly of ...
MISSION® siRNA Universal Negative Controls are an essential component to any siRNA experiment. Using a Negative siRNA control allows the researcher to create a baseline for mRNA knockdown efficiency. MISSION siRNA Universal Negative Controls have been tested in human, rat, and mouse cells. All have carefully designed to have no homology to known gene sequences.Benefits of MISSION Universal Negative siRNA Control:Universal controls designed for use in a wide variety of species including Human, Mouse, Rat, Bovine, Pig, Chicken, Sheep, and CHO cells.Novel design method used to ensure no homology to all Mature and Predicted RefSeq mRNA sequences.Contain no known immune-response motifs.1The unconjugated controls were validated with Agilent 40K human gene arrays to ensure no significant nonspecific gene interactions.Functionally validated to show no immune response via qRT PCRTwo different Universal Negative Control
Due to reasons discussed in the previous post, almost all of these programs are Direct RNAi programs, i.e. the siRNA is administered close to the diseased site. All except for Acuitys program are also with siRNAs that have been chemically modified (to enhance stability etc), and it remains to be seen whether Acuitys strategy to plunge into the clinic first was a wise one. Sirna Therapeutics soon followed suit, but I think took the right decision to invest time to carefully think about how to position their potential product in the more and more crowded AMD market. Sirna Therapeutics was also the first public company based on RNAi. Formerly known as Ribozyme Pharmaceuticals, they leveraged their experience with RNAs to build an operation that had all the tools to to build a decent IP portfolio and quickly enter the clinic. This IP portfolio is mostly based on chemistry and targeting many genes one by one such that it could claim exclusivity for targeting those genes with RNAi. It remains to be ...
RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the CYP3A4 expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with CYP3A4 gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced CYP3A4 mRNA levels by 65% and protein expression levels by 75%. All these siRNAs did not affect the expression of CYP3A5 at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, ...
Benenson and colleagues engineered a target gene to be sensitive to several different siRNAs of their own design. In the simplest case, they introduced a single siRNA molecule to switch off a target gene that encoded a fluorescent protein. In more complex cases, a pair of siRNAs or either of two siRNAs switched off another target gene, which in turn switched off a gene for a fluorescent protein. To make sure the system worked as intended, the researchers based their siRNAs on those of other species, they report in a paper published online today by Nature Biotechnology ...
Download Free Full-Text of an article RORC2 GENE SILENCING IN HUMAN TH17 CELLS BY SIRNA: DESIGN AND EVALUATION OF HIGHLY EFFICIENT SIRNA
Anew detection mechanism for the control of successful siRNA delivery to cells or tissue is introduced using a siRNA-based probe that is capable of inducing a ...
PlanningOperationsProduct ManagementProductionPublic RelationsResearchSalesOther Yes, I need to discuss cells from Adweek about programs, formats and entries that they are may regulate of siRNA Design: Methods and Protocols to me. You emit together randomized to this siRNA Design: Methods. puncture us to find up to siRNA realize as completed to this selection.
siRNA transfection is a powerful tool used to understand the mechanisms of gene regulation and molecular pathways. The following 10 tips will help you to optimize your siRNA transfection.
RNA interference (RNAi) is a powerful tool in the study of gene function. We added poly(A) tails to the 3 ends of siRNA antisense strands by in vitro..
Acquired by Silicon Image Anchor Bay Technologies is developing a new class of components and systems, based on high-performance Digital Video and Audio format-conversion technologies, for the Digital A/V market.. ...
Transcription factor; can act both as activator and as repressor. Binds the 5-CACCC-3 core sequence. Binds to the promoter region of its own gene and can activate its own transcription. Regulates the expression of key transcription factors during embryonic development. Plays an important role in maintaining embryonic stem cells, and in preventing their differentiation. Required for establishing the barrier function of the skin and for postnatal maturation and maintenance of the ocular surface. Involved in the differentiation of epithelial cells and may also function in skeletal and kidney development. Contributes to the down-regulation of p53/TP53 transcription. (Source: UniProt http://www.uniprot.org/uniprot/o43474 ...
Techniques that regulate the level of protein expression by targeting genes at the DNA or RNA level have proven to be powerful strategies in the drive to understand protein expression and function. However, because of their very nature, these techniques are restricted in terms of speed, specificity and reversibility. For instance, these genetic methods of disrupting protein expression can take days to weeks; consequently, cellular and molecular compensation may occur, thereby obscuring expected phenotypes. In addition, as these genetic manipulations result in the eradication of all mRNA splice isoforms, as well as post-translationally modified versions of targeted proteins, these methods lack specificity and are largely limited to studying context-dependent protein function. Finally, the reversibility of these genetic manipulations, including many recently developed on-and-off inducible methods, is relatively slow (being achieved on a timescale of days to weeks) and is incomplete. These ...
SiRNAs exert their biological effect by guiding the degradation of their cognate mRNA sequence, thereby shutting down the corresponding protein production (gene silencing by RNA interference or RNAi). Due to this property, siRNAs are emerging as promising therapeutic agents for the treatment of inherited and acquired diseases, as well as research tools for the elucidation of gene function in both health and disease. Because of their lethality and prevalence, lung diseases have attracted particular attention as targets of siRNA-mediated cures. In addition, lung is accessible to therapeutic agents via multiple routes, e.g., through the nose and the mouth, thus obviating the need for targeting and making it an appealing target for RNAi-based therapeutic strategies. The clinical success of siRNA-mediated interventions critically depends upon the safety and efficacy of the delivery methods and agents. Delivery of siRNAs relevant to lung diseases has been attempted through multiple routes and using various
2345 Small interfering RNA (siRNA) oligonucleotides have been shown to be potent mediators of RNA interference (RNAi) that induce gene silencing with a high degree of sequence specificity. This has resulted in a rapid shift from antisense and ribozymes to siRNA for gene knockdown studies, and thus sparked strong interest in their use as therapeutics. Therapeutic use of siRNA, however, requires both improved biological stability and intracellular targeting. Thus far, gene inhibition by synthetic siRNA following intravenous administration has been limited to high pressure administration yielding primary effects in liver or using lipoplexes yielding primary effects in lung, in both cases via poorly understood mechanisms not readily adaptable to any other tissue and lacking clinical applicability. Recent work with aqueous siRNA administered parenterally by many routes found it could elicit effects on implanted tumors but without correlation to tumor exposure or tumor gene inhibition, leading the ...
Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2-8 of either siRNA strand counting from the 5-end) and complementary sequences in the 3UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found ...
Four pre-designed shRNA constructs targeting caspase 8, apoptosis-related cysteine peptidase (CASP8), transcript variant E and one scrambled control. Each shRNA construct is driven by the U6 promoter and contains a GFP reporter.
Four pre-designed shRNA constructs targeting chromosome 3 open reading frame 58 (C3orf58), transcript variant 2 and one scrambled control. Each shRNA construct is driven by the U6 promoter and contains a GFP reporter.
TY - JOUR. T1 - RNA interference technology used for the study of aquatic virus infections. AU - Reshi, Mohammad Latif. AU - Wu, Jen Leih. AU - Wang, Hao Ven. AU - Hong, Jiann Ruey. N1 - Funding Information: This work was supported by grants NSC 97-2313-B-006-004-MY3 and NCS 102-3011-P-006-002 , awarded to Dr. Jiann-Ruey Hong from the National Science Council, Taiwan, Republic of China.. PY - 2014/9. Y1 - 2014/9. N2 - Aquaculture is one of the most important economic activities in Asia and is presently the fastest growing sector of food production in the world. Explosive increases in global fish farming have been accompanied by an increase in viral diseases. Viral infections are responsible for huge economic losses in fish farming, and control of these viral diseases in aquaculture remains a serious challenge. Recent advances in biotechnology have had a significant impact on disease reduction in aquaculture. RNAi is one of the most important technological breakthroughs in modern biology, ...
In this study, we developed anionic polymer-coated liposome/siRNA complexes (lipoplexes) with chondroitin sulfate C (CS), poly-l-glutamic acid (PGA) and poly-aspartic acid (PAA) for siRNA delivery by intravenous injection, and evaluated the biodistribution and gene silencing effect in mice. The sizes of CS-, PGA- and PAA-coated lipoplexes were about 200?nm and their ?-potentials were negative. CS-, PGA- and PAA-coated lipoplexes did not induce agglutination after mixing with erythrocytes. In terms of biodistribution, siRNAs after intravenous administration of cationic lipoplexes were largely observed in the lungs, but those of CS-, PGA- and PAA-coated lipoplexes were in both the liver and the kidneys, indicating that siRNA might be partially released from the anionic polymer-coated lipoplexes in the blood circulation and accumulate in the kidney, although the lipoplexes can prevent the agglutination with blood components. To increase the association between siRNA and cationic liposome, we used ...
Author Summary RNA interference is a gene regulatory system in which small RNA molecules turn off genes that have similar sequences to the small RNAs. This has become a powerful tool because a researcher can use RNA interference to turn off any gene of interest in order to test its function. There is great interest in identifying the genes required for the RNA interference pathway, and one approach to identifying such genes has been to use RNA interference to turn off potential RNA interference genes and to ask whether RNA interference still functions when these genes are turned off. The goal of our report is to ask how it is possible for RNA interference to turn itself off, using a mathematical model of the system. The results show that RNA interference cannot turn itself off if the RNA interference pathway is too effective to start with, so that experiments in which RNA interference acts on itself will only work in systems having a low efficiency. The results of our model suggest possible ways to
Dicerna Pharmaceuticals, Inc. (NASDAQ:DRNA) , a leader in the development of RNAi therapeutics, today announced it is expanding its ongoing Phase 1 study of DCR-MYC in solid tumors, multiple myeloma, or lymphoma to include a cohort of patients with pancreatic neuroendocrine tumors (PNETs) following early signs of clinical and metabolic response and tumor shrinkage in PNET patients. DCR-MYC is an investigational Dicer substrate short-interfering RNA (DsiRNA) therapeutic targeting the MYC oncogene and the first MYC-targeting short-interfering RNA (siRNA) to enter clinical trials. Based on the clinical activity seen in two out of three patients with PNET, evidence of a complete metabolic response (based on FDG-PET) and a partial response (based on RECIST criteria), as well as published evidence on the role of MYC in growth and maintenance of PNET tumors, we are adding a PNET expansion cohort in the ongoing Phase 1 study, said Pankaj Bhargava, M.D., chief medical officer of Dicerna. Most patients ...
Transposable elements (TEs) are major structural components of eukaryotic genomes; however, mobilization of TEs generally has negative effects on the host genome. To counteract this threat, host cells have evolved genetic and epigenetic mechanisms that keep TEs silenced. One such mechanism involves the Piwi-piRNA complex, which represses TEs in animal gonads either by cleaving TE transcripts in the cytoplasm or by directing specific chromatin modifications at TE loci in the nucleus. Most Piwi-interacting RNAs (piRNAs) are derived from genomic piRNA clusters. There has been remarkable progress in our understanding of the mechanisms underlying piRNA biogenesis. However, little is known about how a specific locus in the genome is converted into a piRNA-producing site. In this review, we will discuss a possible link between chromatin boundaries and piRNA cluster formation.
Specific gene knock-down in post-natal cells by siRNA molecules has immense potential as a research and therapeutic tool. While direct effects have been clearly demonstrated in siRNA-transduced cells, we investigated a possible bystander effect whereby siRNAs transfer between transduced and non-transduced (NT) primary neonatal rat ventricular myocytes (NRVMs) via gap junctions. To enable siRNA expression in NRVMs, lentiviral vectors (LV) encoding short-hairpin (sh)RNAs, which are processed to siRNAs, were generated. Two populations of LV-transduced NRVMs were produced; one expressing GFP and the other co-expressing a second reporter plus a siRNA designed to knock-down GFP. Following 7 days co-culture of these populations, flow cytometry revealed a 35% reduction in the mean GFP fluorescence intensity and real-time PCR confirmed GFP mRNA knock-down. To explore dependence of this effect on gap junctions, we co-transduced cells with a LV encoding a dominant-negative connexin43 mutant (Cx43Δ) as a ...
The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin. To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The
Eukaryotic cells express a complex layer of noncoding RNAs. An intriguing family of regulatory RNAs includes transcripts from the opposite strand of protein coding genes, so called natural antisense transcripts (NATs). Here, we test the hypothesis that antisense transcription triggers RNA interference and gives rise to endogenous short RNAs (endo-siRNAs). We used cloned human embryonic kidney cells (HEK293) followed by short RNAseq to investigate the small genic RNA transcriptome. 378 genes gave rise to short RNA reads that mapped to exons of RefSeq genes. The length profile of short RNAs showed a broad peak of 20-24 nucleotides, indicative of endo-siRNAs. Collapsed reads mapped predominantly to the first and the last exon of genes (74%). RNAs reads were intersected with sequences occupied by RNAPII or bound to Argonaute (AGO1 by crosslinking, ligation, and sequencing of hybrids, CLASH). In the first exon, 94% of the reads correlated with RNAPII occupancy with an average density of 130 (relative units);
Molecular Plant-Microbe Interactions 26:617-625...Jang-Kyun Seo,1 Jianguo Wu,2 Yifan Lii,1 Yi Li,2 and Hailing Jin1...© 2013 The American Phytopathological Society...Small RNAs regulate a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity, in a sequence-specific manner. Accumulating evidence reveals that host endogenous small RNAs and small RNA pathway components play important roles in plan...
Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this siRNA omics approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to
Goodwin Procter associate Daniel Wilson looks into patenting strategies for a powerful new tool for treating disease as well as for creating models of disease.
piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the
Signal-mediated amplification of RNA technology (SMART) is a novel isothermal amplification technology that uses a three-way junction (3WJ) structure to
To make an lentiviral shRNA construct, we only need to know the RefSeq number. We typically design and clone 3-5 constructs that target your transcript. Our proprietary shRNA design delivers approximately 70% shRNAs that generate ,70% transcript knockdown in standard cell lines as measured by qRT-PCR so it is likely that at least 2 of 3 or 3 of 5 constructs will be effective.. We make the inserst, clone them, and sequence the constructs to verify correct construction. You receive several of our best-designed constructs to test and characterize to see which best matches your experimental needs. We can provide the plasmid constructs, or you have the option to also package the constructs as ready-to-transduce lentiviral particles.. We do not guarantee this level for any specific target, and the percentage of highly effective shRNAs will vary from target to target.. ...
Attempts to target mutant KRAS have been unsuccessful. Most of the current therapeutic approaches are indirect, mainly via inhibiting KRAS down-stream signaling, which have been marginally successful. Here we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2), a HECT-type ubiquitin ligase (E3) as a critical regulator of mutant KRAS protein stability. We show that the loss of SMURF2 either by si-/sh-RNA mediated gene silencing or by overexpression of a catalytically inactive SMURF2 Cys716Ala (CA) mutant, can cause lysosome-mediated KRAS degradation; whereas, overexpression of wild type SMURF2 enhances KRAS protein stability. Most importantly, we found that mutant KRAS protein is more susceptible to SMURF2 alterations in that mutant protein half-life decreased from ,12h in control siRNA-treated cells to ,3h with Smurf2 siRNA treatment, whereas only marginal differences were noted for wild-type KRAS protein upon similar treatments. Importantly, this loss of mutant KRAS ...
To further test the vector system, we designed both a synthetic siRNA and a pSUPER vector that target the same 19-nt sequence in theCDC20 transcript. As for CDH1, efficient suppression of endogenous CDC20 expression was achieved with both synthetic siRNA and with pSUPER-CDC20 (Fig. 2B). To measure the level of gene suppression accurately by the pSUPER system, we designed a construct to target polo like kinase-1 (PLK1). Introduction of pSUPER-PLK1 led to a significant decrease in PLK1 protein levels and a reduction in PLK1 kinase activity by a factor of 10 [Supplementary fig. 1A (4)]. To date, we were successful in knocking down the expression of more than 10 genes for which we designed a pSUPER siRNA vector, highlighting the efficiency with which genes can be targeted using this vector (6).. Next, we asked whether suppression of gene expression by the pSUPER vector is sufficient to affect cellular physiology. We designed a construct to knockdown p53, a transcription factor that is stabilized ...
RNAi refers to dsRNA-induced gene silencing, a cellular process that degrades RNA homologous to one strand of the dsRNA [1, 2]. The intermediates of long dsRNA-initiated RNAi are double-stranded small interfering RNAs (siRNA), typically 21-23 nucleotide (nt) long. The siRNAs, when introduced into cells, can be used to silence genes in mammalian systems where long dsRNAs prompt protein kinase R (PKR), RNase L, and interferon activities that result in non-specific RNA degradation and general shutdown of protein synthesis [3]. siRNAs can either be chemically synthesized then directly transfected into cells or can be generated inside the cell by introducing vectors that express short-hairpin RNA (shRNA) precursors of siRNAs. The process of shRNA into functional siRNA involves cellular RNAi machinery that naturally process genome encoded microRNAs (miRNA) that are responsible for cellular regulation of gene expression by modulating mRNA stability, translation, and chromatin structures ...
RNA interference (RNAi) is an incredible revolution in the field of functional genomics, a breakthrough in plant molecular genetics. This technology will generate enormous potential for engineering control of gene expres-sion. The success of managing biotic stress using RNAi technology will prove to be biologically and environmentally safe. It is therapeutic in approach as the resistance induced by RNAi is triggered by ds RNA that results in silencing of specific genes before being translated in a homology dependent manner. Over the time, RNAi is significantly proving it as one of the most promiscent management strategy which eliminates certain risks associated with the development of transgenic plants. This review gives an insight into the probability of management of plant diseases caused by various biotic agents viz. fungi, bacteria and viruses using RNA interference technique and host-pathogen related targeted sites ...
Acute myeloid leukemia (AML) with an NPM1 mutation (NPMc+) has a distinct gene expression signature and displays molecular abnormalities similar to mixed lineage leukemia (MLL), including aberrant expression of the PBX3 and HOXA gene cluster. However, it is unclear if the aberrant expression of PBX3 and HOXA is essential for the survival of NPM1-mutated leukemic cells. Methods: Using the gene expression profiling of TCGA and E-MTAB-3444 datasets, we screened for high co-expression of PBX3 and HOXA9 in NPMc+ leukemia patients. We performed NPMc+ depletion and overexpression experiments to examine aberrant H3K79 methylation through epigenetic regulation. Through RNA interference technology and small-molecule inhibitor treatment, we evaluated the effect of methyl-modified H3K79 on cell survival and explored the possible underlying mechanism. Results: We showed that NPMc+ increased the expression of PBX3 and HOXA9, which are both poor prognosis indicators in AML. High PBX3 and HOXA9 expression was ...
The Human siGENOME RTF G Protein-Coupled Receptor (GPCR) siRNA Library includes SMARTpool siRNA reagents targeting genes encoding GPCRs, transmembrane proteins required for the transduction of extracellular signals from a wide variety of ligands, including neurotransmitters, hormones, and other small molecules. This siRNA Library does not include olefactory or taste receptors.. RTF siRNA libraries are provided as multiple single-use plate sets - just rehydrate and add cells. This unique pre-plated format reduces hands-on time for faster screening results.. siGENOME siRNAs are designed with the proprietary SMARTselection design algorithm for high-efficiency, guaranteed silencing. They also incorporate rational strand bias with application of ON-TARGET modifications to optimize antisense strand loading into RISC for effective target knockdown.. ...
CREB activation and CREB-dependent signaling pathways are crucial for neuronal survival. The term ICER (inducible cAMP early repressor) refers to four protein isoforms that are all endogenous, inducible antagonists of CREB. It was previously shown, that all 4 ICER isoforms are induced upon pro-apoptotic treatment, and also that each of them separately evokes neuronal cell death in cortical culture transfected with these genes. The ICER proteins are believed to be strong repressors of Immediate Early Genes, which are involved in cell response to inter- and/or intra-cellular signals. Herein, we have applied the siRNA approach to silence ICER expression. Because ICERs are members of CREM family of proteins, sharing with them the gene sequence, only the small unique region for ICER was selected to design ICER-directed, specifi c siRNA. Indeed, we obtained functional siRNA capable of blocking ICERs but not affecting CREM proteins. With this tool, we have investigated if the ICERs silencing protects ...
Previous shRNAs to CCR5 identified using conventional commercial algorithms showed cytotoxicity when expressed using the highly active U6 pol III promoter in primary human peripheral blood derived mononuclear cells. Expression using the lower activity H1 promoter significantly reduced toxicity, but all shRNAs also reduced RNAi activity. In an effort to identify shRNAs that were both potent and non-cytotoxic, we created a shRNA library representing all potential CCR5 20 to 22-nucleotide shRNA sequences expressed using an H1 promoter and screened this library for downregulation of CCR5. We identified one potent CCR5 shRNA that was also non-cytotoxic when expressed at a low level with the H1 promoter. We characterized this shRNA in regards to its function and structure. This shRNA was unique that the use of commercial and published algorithms to predict effective siRNA sequences did not result in identification of the same shRNA. We found that this shRNA could induce sequence specific reduction of ...
riboxxFECT is a reagent dedicated for transfection of all formats of siRNA, microRNA mimics and RNA in primary cells, adherent cells or cells in suspension.
siSTABLE GAPD Control siRNA is a validated positive control, guaranteed to silence GAPD in mouse cells. This control siRNA is chemically modified to significantly extend siRNA stability and is recommended for use as a positive control in experiments using siSTABLE modified siRNA against a target gene or where increased stability of the control is desired.. Also known as glyceraldehyde-3-phosphate dehydrogenase or GAPDH, GAPD is an important enzyme in carbohydrate metabolism that is well conserved across the animal kingdom. This gene is abundantly expressed in most cells, and because it is non-essential, knockdown of the corresponding mRNA does not affect cell viability. Targets accession number NM_001001303.. ...
BioAssay record AID 506767 submitted by ChEMBL: Binding affinity to SAP145 in SAP145-targeting siRNA-treated human HeLa cells at 1 uM by fluorescent microscopy.
Page contains details about polymer/DSPC/cholesterol/PEG lipid/siRNA nanoparticles . It has composition images, properties, Characterization methods, synthesis, applications and reference articles : nano.nature.com
Page contains details about PEG-ECO/siRNA nanoparticles . It has composition images, properties, Characterization methods, synthesis, applications and reference articles : nano.nature.com
Development of efficient carriers for small interfering RNA (siRNA) delivery and validation tools for assessing in vivo RNA interference (RNAi) efficiency is crucial to advance RNAi-based therapeutics to the clinic. Here, acid-degradable ketalized linear polyethylenimine (KL-PEI) designed for efficient, stim
The Gates Foundation is backing a biotechnology companys early development of antibodies to treat human immunodeficiency virus or HIV based on RNA.
Looking for a powerful and versatile DNA and siRNA transfection reagent? Try jetPRIME to obtain efficient and reliable scientific results! Request your jetPRIME sample immediately!
Detail záznamu - Paramutation of tobacco transgenes by small RNA-mediated transcriptional gene silencing - Detail záznamu - Knihovna Akademie věd České republiky
...By Genospectra Inc. 6519 Dumbarton Circle Fremont CA 94555. ... ... A critical step in siRNA-mediated gene expression knockdown st... INTRODUCTION There are many sources of exp...,Measuring,siRNA-mediated,knockdown,of,IL-8,mRNA,biological,advanced biology technology,biology laboratory technology,biology device technology,latest biology technology
RNA interference (RNAi) is a post-transcriptional/transcriptional gene silencing mechanism conserved from fungi to humans. In RNAi pathways, small non-coding RN...
Though ZNF 746 known as Parkin-interacting substrate (PARIS) was reported to repress PGC-1α and its target gene NRF-1 leading to the neurodegeneration in Parkinsons disease, its function in tumorigenesis has not been investigated until now. Thus, in the present study, the role of ZNF746 was investigated in the invasion and epithelial to mesenchymal transition (EMT) in ZNF746 overexpressed H460 non-small cell lung cancer (NSCLC) cells. Invasion assay showed that inhibition of ZNF 746 using siRNA transfection method inhibited the invasion of H460 cells using Boyden chamber. Real-time quantitative RT-PCR (RT-qPCR) revealed that the silencing of ZNF 746 attenuated the expression of matrix metalloproteinase (MMP) 1, MMP2 and MMP 9, but not MMP7 in H460 cells. Immunoblotting assay revealed that the expressions of E-cadherin of epithelial phenotype were up-regulated, while Slug was down-regulated in ZNF746 siRNA transfected H460 cells. Consistently, mRNA expression of E-cadherin was up-regulated ...
We have led the way in the development of what has been hailed as a major breakthrough in molecular biology: silencing gene expression by RNA interference (RNAi). CSIROs RNAi gene silencing technology is enabling researchers around the world to protect plants and animals from diseases, and to develop new plant varieties with beneficial attributes.
Supplementary MaterialsFigure S1: The mRNA level in C666-1 and LMP1-transfected cells. cells had been transfected with miR-21 mimics (A) or miR-21 inhibitor (B) or their scrambled control (Scr) and harvested to detect the appearance of miR-21, Fas-L and PDCD4. The fold adjustments had been in accordance with the untreated handles (UC), to which a worth of just one 1.0 was assigned.(TIF) pone.0078355.s003.tif (661K) GUID:?A2E56097-40A0-4C00-A1DF-B3719F9ED900 Figure S4: The result of knocking down LMP1 in CNE2/LMP1 cells in the expression of miR-21, PDCD4 and Fas-L. CNE2/LMP1 cells had been transfected with siLMP1 or control Endoxifen E-isomer hydrochloride siRNA (NC) for 48 h.The cells was harvested and analyzed for LMP1 then, miR-21, PDCD4 and Fas-L by qRT-PCR, and the expression levels in control siRNA transfected cells was set to1. (TIF) pone.0078355.s004.tif (455K) GUID:?C1A19F4B-5588-4974-AD2F-5E973554FEB6 Physique S5: The expression of PDCD4 and Fas-L in response to cisplatin treatment. ...
Dr. Josephine Lai (Professor of Pharmacology, University of Arizona) is a pioneer in developing experimental designs and methods for delivering siRNA to the CNS for gene expression analysis.She and her team have documented these in the publication ...
Genome-wide profiling and functional analyses reveal a network of heterochromatin and small RNA factors that silences repetitive elements and prevents genotoxic stress to ensure fertility.
DECIPHER libraries are barcoded lentiviral shRNA libraries optimized for RNAi Genetic Screens in pooled format deposited by Chenchik and Frangou.
Figure 3. EV Shuttles deliver functional siRNA that can knockdown protein expression in recipient cells. Human HEK293 EV Shuttles were loaded with either an anti-CD81 siRNA or a non-targeting siRNA (NT) and then added to Mouse RAWS 264.7 cells. The cell lysates were collected after 18 hours for Western blot analysis of CD81 protein expression. EV Shuttles carrying the anti-CD81 siRNA were able to reduce CD81 protein expression in the target cells by at least 50% compared to EV Shuttles carrying the NT control siRNA.. EV Shuttles can be used as a non-viral method for generating stable cell lines Because plasmids can be transfected into exosomes with the EV Shuttle System, EV Shuttles can be used to generate stable cell lines without the use of viruses. Here we show an example using our non-viral ...
Alnylam Pharmaceuticals, Inc. (Nasdaq:ALNY), the leading RNAi therapeutics company, today announced that the Company presented new pre-clinical data highlighting its next generation
An arrayed collection of siRNA reagents for RNAi screening, targeting human F-box and SOCS box E3 ubiquitin ligases. Accell siRNA permits RNAi screening in difficult-to-transfect cells with no transfection reagent, virus, or electroporation.
An arrayed siRNA collection targeting human genes involved in cell cycle regulation. siGENOME siRNA is a cost-effective choice for RNAi screening. Available as SMARTpool or 4 individual siRNA reagents.