Journal articles: 'Soaking RNAi'

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Weiner, Alan. "Soaking up RNAi." Molecular Cell 12, no. 3 (September 2003): 535–36. http://dx.doi.org/10.1016/s1097-2765(03)00352-6.

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Arshad, Fiza, Arvind Sharma, Charleen Lu, and Monika Gulia-Nuss. "RNAi by Soaking Aedes aegypti Pupae in dsRNA." Insects 12, no. 7 (July 13, 2021): 634. http://dx.doi.org/10.3390/insects12070634.

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RNA-interference (RNAi) is a standard technique for functional genomics in adult mosquitoes. However, RNAi in immature, aquatic mosquito stages has been challenging. Several studies have shown successful larval RNAi, usually in combination with a carrier molecule. Except for one study in malaria mosquito, Anopheles gambiae, none of the previous studies has explored RNAi in mosquito pupae. Even in the study that used RNAi in pupae, double stranded RNA (dsRNA) was introduced by microinjection. Here, we describe a successful method by soaking pupae in water containing dsRNA without any carrier or osmotic challenge. The knockdown persisted into adulthood. We expect that this simple procedure will be useful in the functional analysis of genes that highly express in pupae or newly emerged adults.

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LILLEY, C. J., L. J. DAVIES, and P. E. URWIN. "RNA interference in plant parasitic nematodes: a summary of the current status." Parasitology 139, no. 5 (January 5, 2012): 630–40. http://dx.doi.org/10.1017/s0031182011002071.

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SUMMARYRNA interference (RNAi) has emerged as an invaluable gene-silencing tool for functional analysis in a wide variety of organisms, particularly the free-living model nematode Caenorhabditis elegans. An increasing number of studies have now described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when nematodes take up double stranded RNA (dsRNA) or short interfering RNAs (siRNAs) that elicit a systemic RNAi response. Despite many successful reports, there is still poor understanding of the range of factors that influence optimal gene silencing. Recent in vitro studies have highlighted significant variations in the RNAi phenotype that can occur with different dsRNA concentrations, construct size and duration of soaking. Discrepancies in methodology thwart efforts to reliably compare the efficacy of RNAi between different nematodes or target tissues. Nevertheless, RNAi has become an established experimental tool for plant parasitic nematodes and also offers the prospect of being developed into a novel control strategy when delivered from transgenic plants.

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McMaster, Steven, Susan McKinney, Aaron Maule, Michael Kimber, Colin Fleming, Philip Donnelly, and Michael Johnston. "Getting to the root of neuronal signalling in plant-parasitic nematodes using RNA interference." Nematology 9, no. 3 (2007): 301–15. http://dx.doi.org/10.1163/156854107781351981.

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AbstractA variety of genes expressed in preparasitic second-stage juveniles (J2) of plant-parasitic nematodes appear to be vulnerable to RNA interference (RNAi) in vitro by coupling double-stranded (ds)RNA soaking with the artificial stimulation of pharyngeal pumping. Also, there is mounting evidence that the in planta generation of nematode-specific double-stranded RNAs (dsRNAs) has real utility in the control of these pests. Although neuronally-expressed genes in Caenorhabditis elegans are commonly refractory to RNAi, we have discovered that neuronally-expressed genes in plant-parasitic nematodes are highly susceptible to RNAi and that silencing can be induced by simple soaking procedures without the need for pharyngeal stimulation. Since most front-line anthelmintics that are used for the control of nematode parasites of animals and humans act to disrupt neuromuscular coordination, we argue that intercellular signalling processes associated with neurons have much appeal as targets for transgenic plant-based control strategies for plant-parasitic nematodes. FMRFamide-like peptides (FLPs) are a large family of neuropeptides which are intimately associated with neuromuscular regulation, and our studies on flp gene function in plant-parasitic nematodes have revealed that their expression is central to coordinated locomotory activities. We propose that the high level of conservation in nervous systems across nematodes coupled with the RNAi-susceptibility of neuronally-expressed genes in plant-parasitic nematodes provides a valuable research tool which could be used to interrogate neuronal signalling processes in nematodes.

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Li, Chengjian, and Phillip D. Zamore. "RNAi in Drosophila S2 Cells by dsRNA Soaking." Cold Spring Harbor Protocols 2019, no. 3 (March 2019): pdb.prot097477. http://dx.doi.org/10.1101/pdb.prot097477.

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Zhou, R., S. Mohr, G. J. Hannon, and N. Perrimon. "Inducing RNAi in Drosophila Cells by Soaking with dsRNA." Cold Spring Harbor Protocols 2014, no. 5 (May 1, 2014): pdb.prot080747. http://dx.doi.org/10.1101/pdb.prot080747.

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ZAWADZKI, J. L., A. C. KOTZE, J. A. FRITZ, N. M. JOHNSON, J. E. HEMSWORTH, B. M. HINES, and C. A. BEHM. "Silencing of essential genes by RNA interference in Haemonchus contortus." Parasitology 139, no. 5 (February 20, 2012): 613–29. http://dx.doi.org/10.1017/s0031182012000121.

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SUMMARYIn this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the ‘essential’ genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.

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Mon, Hiroaki, Zhiqing Li, Isao Kobayashi, Shuichiro Tomita, JaeMan Lee, Hideki Sezutsu, Toshiki Tamura, and Takahiro Kusakabe. "Soaking RNAi inBombyx moriBmN4-SID1 Cells Arrests Cell Cycle Progression." Journal of Insect Science 13, no. 155 (December 2013): 1–7. http://dx.doi.org/10.1673/031.013.15501.

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Zhu, Kun Yan, and Subba Reddy Palli. "Mechanisms, Applications, and Challenges of Insect RNA Interference." Annual Review of Entomology 65, no. 1 (January 7, 2020): 293–311. http://dx.doi.org/10.1146/annurev-ento-011019-025224.

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The RNA interference (RNAi) triggered by short/small interfering RNA (siRNA) was discovered in nematodes and found to function in most living organisms. RNAi has been widely used as a research tool to study gene functions and has shown great potential for the development of novel pest management strategies. RNAi is highly efficient and systemic in coleopterans but highly variable or inefficient in many other insects. Differences in double-stranded RNA (dsRNA) degradation, cellular uptake, inter- and intracellular transports, processing of dsRNA to siRNA, and RNA-induced silencing complex formation influence RNAi efficiency. The basic dsRNA delivery methods include microinjection, feeding, and soaking. To improve dsRNA delivery, various new technologies, including cationic liposome–assisted, nanoparticle-enabled, symbiont-mediated, and plant-mediated deliveries, have been developed. Major challenges to widespread use of RNAi in insect pest management include variable RNAi efficiency among insects, lack of reliable dsRNA delivery methods, off-target and nontarget effects, and potential development of resistance in insect populations.

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Marmonier, Aurélie, Laetitia Perfus-Barbeoch, Corinne Rancurel, Sylvaine Boissinot, Bruno Favery, Gérard Demangeat, and Véronique Brault. "In Vitro Acquisition of Specific Small Interfering RNAs Inhibits the Expression of Some Target Genes in the Plant Ectoparasite Xiphinema index." International Journal of Molecular Sciences 20, no. 13 (July 3, 2019): 3266. http://dx.doi.org/10.3390/ijms20133266.

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Xiphinema index is an important plant parasitic nematode that induces direct damages and specifically transmits the Grapevine fanleaf virus, which is particularly harmful for grapevines. Genomic resources of this nematode species are still limited and no functional gene validation technology is available. RNA interference (RNAi) is a powerful technology to study gene function and here we describe the application of RNAi on several genes in X. index. Soaking the nematodes for 48 h in a suspension containing specific small interfering RNAs resulted in a partial inhibition of the accumulation of some targeted mRNA. However, low reproducible silencing efficiency was observed which could arise from X. index silencing pathway deficiencies. Indeed, essential accustomed proteins for these pathways were not found in the X. index proteome predicted from transcriptomic data. The most reproducible silencing effect was obtained when targeting the piccolo gene potentially involved in endo-exocytosis of synaptic molecules. This represents the first report of gene silencing in a nematode belonging to the Longidoridae family.

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KRAUTZ-PETERSON, GREICE, RITA BHARDWAJ, ZAHRA FAGHIRI, CIBELE A. TARARAM, and PATRICK J. SKELLY. "RNA interference in schistosomes: machinery and methodology." Parasitology 137, no. 3 (September 21, 2009): 485–95. http://dx.doi.org/10.1017/s0031182009991168.

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SUMMARYRNA interference (RNAi) is a potent gene silencing process that is playing an increasingly important role in investigations of gene function in schistosomes. Here we review what is known about the process in these parasites and provide an update on the methodology and machinery of RNAi. Data are presented to demonstrate that: (1) not all schistosome genes can be suppressed to the same extent, using the methods employed here; (2) while there is variation in the level of suppression achieved for one target gene (SmAP) in adult parasites, all individuals exhibit robust (>80%) suppression; (3) short interfering RNAs (siRNAs) can effect suppression when delivered by soaking (and not just via electroporation, as reported previously); (4) Male/female adult pairs need not be separated prior to siRNA delivery by electroporation for effective gene suppression in both genders and (5) electroporation of siRNAs in medium is as efficient as in commercial electroporation buffer. Regarding the machinery of RNAi in schistosomes, a homologue of the C. elegans multi-membrane spanning, RNA importing protein SID-1 is identified in silico. The gene encoding this protein contains 21 exons and spans over 50 kb to potentially encode a 115,556 Mr protein (SmSID-1). These analyses, and a review of the literature, permit us to derive and present here a draft of potential RNAi pathways in schistosomes.

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GELDHOF, P., A. VISSER, D. CLARK, G. SAUNDERS, C. BRITTON, J. GILLEARD, M. BERRIMAN, and D. KNOX. "RNA interference in parasitic helminths: current situation, potential pitfalls and future prospects." Parasitology 134, no. 5 (May 2006): 609–19. http://dx.doi.org/10.1017/s0031182006002071.

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SUMMARYRNA interference (RNAi) has become an invaluable tool for the functional analysis of genes in a wide variety of organisms including the free-living nematode Caenorhabditis elegans. Recently, attempts have been made to apply this technology to parasitic helminths of animals and plants with variable success. Gene knockdown has been reported for Schistosoma mansoni by soaking or electroporating different life-stages in dsRNA. Similar approaches have been tested on parasitic nematodes which clearly showed that, under certain conditions, it was possible to interfere with gene expression. However, despite these successes, the current utility of this technology in parasite research is questionable. First, problems have arisen with the specificity of RNAi. Treatment of the parasites with dsRNA resulted, in many cases, in non-specific effects. Second, the current RNAi methods have a limited efficiency and effects are sometimes difficult to reproduce. This was especially the case in strongylid parasites where only a small number of genes were susceptible to RNAi-mediated gene knockdown. The future application of RNAi in parasite functional genomics will greatly depend on how we can overcome these difficulties. Optimization of the dsRNA delivery methods and in vitro culture conditions will be the major challenges.

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Rosso, M. N., M. P. Dubrana, N. Cimbolini, S. Jaubert, and P. Abad. "Application of RNA Interference to Root-Knot Nematode Genes Encoding Esophageal Gland Proteins." Molecular Plant-Microbe Interactions® 18, no. 7 (July 2005): 615–20. http://dx.doi.org/10.1094/mpmi-18-0615.

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Plant parasitic nematodes have been, so far, refractory to transformation or mutagenesis. The functional analysis of nematode genes relies on the development of reverse genetic tools adapted to these obligate parasites. Here, we describe the application of RNA interference (RNAi) to the root-knot nematode Meloidogyne incognita for the knock-down of two genes expressed in the subventral esophageal glands of the nematode and potentially involved in parasitism, the calreticulin (Mi-crt) and the polygalacturonase (Mi-pg-1) genes. Incubation in 1% resorcinol for 4 h induced double-stranded RNA uptake through the alimentary track of the nematodes and led to up to 92% depletion of Mi-crt transcripts. Timecourse analysis of the silencing showed different temporal patterns for Mi-crt and Mi-pg-1. The silencing of Mi-crt was optimal 20 h after soaking, whereas the silencing of Mi-pg-1 was optimal 44 h after soaking. For the two genes, the silencing effect was highly time-limited, since no transcript depletion was detectable 68 h after soaking.

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Baniya, Anil, Soumi Joseph, Larry Duncan, William Crow, and Tesfamariam Mengistu. "The role of Caenorhabditis elegans sex-determination homologs, Mi-sdc-1 and Mi-tra-1 in Meloidogyne incognita." European Journal of Plant Pathology 161, no. 2 (July 28, 2021): 439–52. http://dx.doi.org/10.1007/s10658-021-02335-3.

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AbstractSex determination is a key developmental event in all organisms. The pathway that regulates sexual fate has been well characterized at the molecular level in the model free-living nematode Caenorhabditis elegans. This study aims to gain a preliminary understanding of sex-determining pathways in a plant-parasitic nematode Meloidogyne incognita, and the extent to which the roles of the sex determination genes are conserved in a hermaphrodite species, C. elegans, and plant-parasitic nematode species, M. incognita. In this study, we targeted two sex-determining orthologues, sdc-1 and tra-1 from M. incognita using RNA interference (RNAi). RNAi was performed by soaking second-stage juveniles of M. incognita in a solution containing dsRNA of either Mi-tra-1or Mi-sdc-1 or both. To determine the effect of RNAi of the target genes, the juveniles treated with the dsRNA were inoculated onto a susceptible cultivar of cowpea grown in a nutrient pouch at 28 °C for 5 weeks. The development of the nematodes was analyzed at different time points during the growth period and compared to untreated controls. Our results showed that neither Mi-sdc-1 nor Mi-tra-1 have a significant role in regulating sexual fate in M. incognita. However, the silencing of Mi-sdc-1 significantly delayed maturity to adult females but did not affect egg production in mature females. In contrast, the downregulation of Mi-tra-1 transcript resulted in a significant reduction in egg production in both single and combinatorial RNAi-treated nematodes. Our results indicate that M. incognita may have adopted a divergent function for Mi-sdc-1 and Mi-tra-1distinct from Caenorhabditis spp. However, Mi-tra-1 might have an essential role in female fecundity in M. incognita and is a promising dsRNA target for root-knot nematode (RKN) management using host-delivered RNAi.

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Xu, Jian, Yudai Nagata, Hiroaki Mon, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, Takahiro Kusakabe, and Jae Man Lee. "Soaking RNAi-mediated modification of Sf9 cells for baculovirus expression system by ectopic expression of Caenorhabditis elegans SID-1." Applied Microbiology and Biotechnology 97, no. 13 (March 7, 2013): 5921–31. http://dx.doi.org/10.1007/s00253-013-4785-1.

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Cheng, Xin-Yue, Su-Ming Dai, Luo Xiao, and Bing-Yan Xie. "Influence of cellulase gene knockdown by dsRNA interference on the development and reproduction of the pine wood nematode, Bursaphelenchus xylophilus." Nematology 12, no. 2 (2010): 225–33. http://dx.doi.org/10.1163/138855409x12469541205044.

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Abstract Cellulase genes are very important for plant-parasitic nematodes to move and feed within their host plants. The pine wood nematode, Bursaphelenchus xylophilus, causes destructive damage by killing pine trees. In this study, by employing dsRNA interference technology, knockdown of a cellulase gene (Bx-eng-1) of B. xylophilus was achieved and the biological effects of RNAi on the nematode were observed. The result showed that, after 24 h soaking, dsRNA of the Bx-eng-1 gene was effectively delivered into the nematode causing a post-transcriptional gene silencing and decrease in cellulase activity. Moreover, the number of F1 generation offspring was reduced significantly when the dsRNA-treated nematodes were cultured on fungal mats. We consider that cellulase is important to B. xylophilus because it not only hydrolyses cellulose of plant cell wall for its parasitism and penetration in host plants, but also influences its feeding, development and propagation on fungal mats.

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BECKMANN, S., and C. G. GREVELDING. "Paving the way for transgenic schistosomes." Parasitology 139, no. 5 (September 6, 2011): 651–68. http://dx.doi.org/10.1017/s0031182011001466.

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SUMMARYIn parasitological research, significant progress has been made with respect to genomics and transcriptomics but transgenic systems for functional gene analyses are mainly restricted to the protozoan field. Gene insertion and knockout strategies can be applied to parasitic protozoa as well as gene silencing by RNA interference (RNAi). By contrast, research on parasitic helminthes still lags behind. Along with the major advances in genome and transcriptome analyses e.g. for schistosomes, methods for the functional characterization of genes of interest are still in their initial phase and have to be elaborated now, at the beginning of the post-genomic era. In this review we will summarize attempts made in the last decade regarding the establishment of protocols to transiently and stably transform or transfect schistosomes. Besides approaches using particle bombardment, electroporation or virus-based infection strateies to introduce DNA constructs into adult and larval schistosome stages to express reporter genes, first approaches have also been made in establishing protocols based on soaking, lipofection, and/or electroporation for RNA interference to silence gene activity. Although in these cases remarkable progress can be seen, the schistosome community eagerly awaits major breakthroughs especially with respect to stable transformation, but also for silencing or knock-down strategies for every schistosome gene of interest.

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Qiu, Xiuwen, Lili Yang, Jianren Ye, Wei Wang, Tiantian Zhao, Hao Hu, and Guixiang Zhou. "Silencing of cyp-33C9 Gene Affects the Reproduction and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus." International Journal of Molecular Sciences 20, no. 18 (September 12, 2019): 4520. http://dx.doi.org/10.3390/ijms20184520.

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Cytochrome P450 genes are very important for plant-parasitic nematodes to reproduce and to metabolize xenobiotic compounds generated by their host plants. The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes very high annual economic losses by killing large numbers of pine trees across Asia and into Europe. In this study, we used RNA interference (RNAi) to analyze the function of the cyp-33C9 gene of PWN. Our results showed that expression of the cyp-33C9 gene was suppressed successfully after soaking nematodes for 24 h in cyp-33C9 double-stranded RNA (dsRNA). The silencing of the cyp-33C9 gene significantly decreased the feeding, reproduction, oviposition and egg hatch of B. xylophilus. Meanwhile, the migration speed of B. xylophilus in Pinus thunbergii was reduced in the early stages when the cyp-33C9 gene was silenced in the nematodes. Moreover, knockdown of the cyp-33C9 gene in B. xylophilus caused a decrease in pathogenicity to pine trees. These results suggest that the cyp-33C9 gene plays an important role in the reproduction and pathogenicity of B. xylophilus. This discovery identified several functions of the cyp-33C9 gene in B. xylophilus and provided useful information for understanding the molecular mechanism behind pine wilt disease caused by PWN.

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Hammond, Scott M. "Soaking up small RNAs." Nature Methods 4, no. 9 (September 2007): 694–95. http://dx.doi.org/10.1038/nmeth0907-694.

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Ketting, René F., Marcel Tijsterman, and Ronald H. A. Plasterk. "Introduction of Double-Stranded RNA inC. elegansby Soaking." Cold Spring Harbor Protocols 2006, no. 1 (January 1, 2006): pdb.prot4316. http://dx.doi.org/10.1101/pdb.prot4316.

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Orii, Hidefumi, Makoto Mochii, and Kenji Watanabe. "A simple "soaking method" for RNA interference in the planarian Dugesia japonica." Development Genes and Evolution 213, no. 3 (March 12, 2003): 138–41. http://dx.doi.org/10.1007/s00427-003-0310-3.

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Inyagwa, Charles Muleke, and Dave Knox. "RNA Interference of the Hemonchus contortus Astacin-like MTP Gene by the Soaking Method." International Journal of Current Microbiology and Applied Sciences 9, no. 4 (April 10, 2020): 1175–84. http://dx.doi.org/10.20546/ijcmas.2020.904.139.

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Wojtas, Magdalena N., and Nicola G. A. Abrescia. "Soaking of DNA into crystals of archaeal RNA polymerase achieved by desalting in droplets." Acta Crystallographica Section F Structural Biology and Crystallization Communications 68, no. 9 (August 31, 2012): 1134–38. http://dx.doi.org/10.1107/s1744309112033507.

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Xu, Jian, Hiroaki Mon, Takahiro Kusakabe, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, Atsushi Masuda, Takumi Mitsudome, and Jae Man Lee. "Establishment of a soaking RNA interference and Bombyx mori nucleopolyhedrovirus (BmNPV)-hypersensitive cell line using Bme21 cell." Applied Microbiology and Biotechnology 97, no. 24 (October 1, 2013): 10435–44. http://dx.doi.org/10.1007/s00253-013-5279-x.

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Ferre-D'Amare, Adrian. "Dehydration and cation replacement dramatically improve crystals of large RNAs." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1149. http://dx.doi.org/10.1107/s2053273314088500.

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Preparation of well-ordered crystals of large RNAs remains a daunting experimental challenge. This probably reflects the relatively undifferentiated molecular surface of folded RNAs (dominated by a regular array of phosphates), the comparatively low free energy of RNA tertiary structure stabilization, and the resulting tendency of RNAs to be conformationally polydisperse. We have found empirically that dehydration and exchange of counterions, can dramatically improve the diffraction properties of some RNA crystals. Two examples from our work are crystals of the glmS ribozyme-riboswitch [1] and of the ternary complex of a T-box riboswitch, its cognate tRNA and an RNA binding protein [2]. Untreated, flash-frozen crystals of the glmS ribozyme diffracted synchrotron X-rays to 3.3 Å resolution. Upon controlled dehydration by soaking into solutions with higher osmolarity, the unit cell contracted by approximately 10%, and diffraction data could be collected that extended to 1.7 Å resolution. Untreated crystals of the T-box ternary complex diffracted X-rays only to 8 Å resolution. A combination of controlled dehydration and exchange of the magnesium an lithium ions needed for crystal growth with strontium (a soft divalent cation), dramatically extended the diffraction limit, allowing the structure to be solved by SAD at 3.2 Å resolution. Because it is a polyanion, RNA is heavily hydrated and surrounded by a diffuse cloud of counterions. It can also site-specifically bind to partially or wholly desolvated metal ions. Hydration and ion binding not only control RNA folding, but also modulate crystallogenesis. Therefore, controlled dehydration and cation exchange are post-crystallization treatments that should be routinely explored for RNA. This work was supported in part by the Intramural Program of the National Heart, Lung and Blood Institute.

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Liu, Bin, Yuhong Zuo, and Thomas A. Steitz. "Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism." Proceedings of the National Academy of Sciences 113, no. 15 (March 28, 2016): 4051–56. http://dx.doi.org/10.1073/pnas.1520555113.

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In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

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Yu, Xiudao, Siddarame Gowda, and Nabil Killiny. "Double-stranded RNA delivery through soaking mediates silencing of the muscle protein 20 and increases mortality to the Asian citrus psyllid, Diaphorina citri." Pest Management Science 73, no. 9 (March 14, 2017): 1846–53. http://dx.doi.org/10.1002/ps.4549.

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Rozovski, Uri, George Calin, Setoyama Tetsirp, Lucilla D'Abundo, David Harris, Ping Li, Zhiming Liu, et al. "Signal Transducer and Activator of Transcription (STAT)-3-Dependent Regulation of Non-Coding RNA Genes in Chronic Lymphocytic Leukemia (CLL)." Blood 120, no. 21 (November 16, 2012): 2886. http://dx.doi.org/10.1182/blood.v120.21.2886.2886.

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Abstract Abstract 2886 Non-coding RNAs regulate the expression of more than 30% of protein-coding genes both at a post-transcriptional and translational level. Although approximately 1000 microRNAs (miRs) have been identified in the human genome, little is known about the mechanisms that regulate miR expression. STAT3 regulates the transcription of miR-21, and miR181b-1, binds to their promoter and induce neoplastic cell transformation (Iliopoulos, Jaeger et al. 2010). Because STAT3 is constitutively activated in CLL cells (Hazan-Halevy, Harris et al. 2010) we sought to investigate how STAT3 affects non-coding RNA gene expression in CLL cells. We transfected peripheral blood CLL cells from 3 different patients with STAT3-shRNA and assessed non-coding RNA levels using a non-coding RNA array containing 2277 human miR probes, 960 from ultra-conserved genes and 3540 of long non-coding RNAs. When compared to transfection control, 152 probes from 78 non-coding RNA genes were differentially expressed (134 down-regulated and 18 up-regulated), suggesting that STAT3 affects the non-coding RNA network in CLL cells. Supervised clustering analysis was used to select genes for validation. By using quantitative RT-PCR we validated our gene array analysis. Similar to the data obtained by the non-coding RNA array, we found that transfection of CLL cells with STAT3- down-regulated the levels of miR-21, miR-155, and miR-320b. Binding site prediction programs and ChIP-seq data embedded in the UCSC genome browser determined that in 5 of 7 genes, down-regulated by STAT3-shRNA transfection, were either putative or experimentally confirmed STAT3-binding sites, indication that STAT3 directly regulates the transcription of those miRs. It has been shown that the interaction between miRs and single stranded RNA is dependent on base pairing in a seed region at positions 2 to 8. High levels of 4.8kb single stranded STAT3 RNA transcripts, present in CLL cells, provide a substrate for such paring. Therefore, we assumed that STAT3 functions as a “RNA sponge” soaking up miRs and altering their effective levels and function. To test this hypothesis we used the pattern-based RNA22 algorithm and identified potential miR targets. We than calculated the energy that would be released if the corresponding RNA/RNA complexes are saturated. We found that the energy released from binding of miRs to STAT3 sequences would be higher than energy released from binding to a random sequence with same length and base content suggesting that STAT3 “sponges out” miRs in a sequence specific manner. Thus, CLL cells are characterized by an ongoing interaction between STAT3-mediated transcriptional regulation of non-coding RNA and miR-mediated translational regulation of coding genes. Disclosures: No relevant conflicts of interest to declare.

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Uri, Rozovski, George Calin, Tetsoro Setoyama, Abundu Lucilla, Harris David, Ping Li, Lui Zhiming, et al. "Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells." Blood 122, no. 21 (November 15, 2013): 4152. http://dx.doi.org/10.1182/blood.v122.21.4152.4152.

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Abstract MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL. Disclosures: No relevant conflicts of interest to declare.

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Chen, Siye. "The Use of RNA Interference in Enhancing Plant Resistance against Nematodes." Journal of Botanical Research 2, no. 1 (April 30, 2020). http://dx.doi.org/10.30564/jrb.v2i1.1759.

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Plant-parasitic nematodes caused severe yield loss in major crops all over the world. The most wild-used strategies to combat the nematodes is the chamical nematicides, but the overuse of synthetic nematicides threaten sustainable agriculture development. Other strategies, like resistance cultivars and crop rotation, have limited efficiency. Thus, the utilization of molecular biotechnology like RNA interference (RNAi) would be one of the alternative ways to enhance plant resistance against nematodes. RNAi has already used as a tool for gene functional analysis in a wide range of species, especially in the non-parasitic nematode, Caenorhabtidis elegans. In plant-parasitic nematodes, RNAi is induced by soaking nematodes with double-strand RNA(dsRNA) solution mixed with neurostimulants, which is called in vitro RNAi delivery method. In another way around, in planta RNAi method, which is Host-mediated RNAi approach also showed a great success in conferring the resistance against root-knock nematodes. Two main advantages of RNAi-based transgenics are RNAi technology do not produce any functional foreign proteins and it target organisms in a sequence-specific way. Even though the development of RNAi-based transgenics against plant-parasitic nematodes is still in the initial phase, it offers the prospect into a novel nematode control strategy in the future.

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Munawar, Kashif, Azzam M. Alahmed, and Sayed M. S. Khalil. "Delivery Methods for RNAi in Mosquito Larvae." Journal of Insect Science 20, no. 4 (July 1, 2020). http://dx.doi.org/10.1093/jisesa/ieaa074.

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Abstract Mosquito-transmitted diseases pose a threat for a great portion of the world population. Chemical insecticides are the main tool for mosquito control. Heavy dependence on chemicals created several problems such as resistance development in many mosquito species, environmental effects, and human health issues. Other tools for mosquito control were developed and used in some parts of the world. Ribonucleic acid interference (RNAi) is a reverse genetic mechanism that was recently introduced as a new tool for pest control. Regarding mosquito, RNAi was used to study gene function and to discover genes that can be used as targets for control purposes. Several delivery methods are used to induce RNAi in mosquito larvae. Some methods such as injection and soaking are used routinely in RNAi research but have no application in the field. Other methods such as nanoparticles and microbes have some characteristics that make them good candidates for field application. In this report, we will focus on delivery methods for RNAi in mosquito larvae and will give examples for each method.

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Khalil, Sayed M. S., Kashif Munawar, Azzam M. Alahmed, and Ahmed M. A. Mohammed. "RNAi-Mediated Screening of Selected Target Genes Against Culex quinquefasciatus (Diptera: Culicidae)." Journal of Medical Entomology, July 1, 2021. http://dx.doi.org/10.1093/jme/tjab114.

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Abstract Culex quinquefasciatus, a member of the Culex pipiens complex, is widespread in Saudi Arabia and other parts of the world. It is a vector for lymphatic filariasis, Rift Valley fever, and West Nile virus. Studies have shown the deleterious effect of RNA interference (RNAi)-mediated knockdown of various lethal genes in model and agricultural pest insects. RNAi was proposed as a tool for mosquito control with a focus on Aedes aegypti and Anopheles gambiae. In this study, we examined the effect of RNAi of selected target genes on both larval mortality and adult emergence of Cx. quinquefasciatus through two delivery methods: soaking and nanoparticles. Ten candidate genes were selected for RNAi based on their known lethal effect in other insects. Disruption of three genes, chitin synthase-1, inhibitor of apoptosis 1, and vacuolar adenosine triphosphatase, resulted in the highest mortality among the selected genes using the two treatment methods. Silencing the other seven genes resulted in a medium to low mortality in both assays. These three genes are also active against a wide range of insects and could be used for RNAi-based mosquito control in the future.

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Khodakova, Anastasia S., Daniela Vidal Vilchis, Dana Blackburn, Ferdinand Amanor, and Buck S. Samuel. "Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation." G3 Genes|Genomes|Genetics 11, no. 7 (April 19, 2021). http://dx.doi.org/10.1093/g3journal/jkab123.

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Abstract The free-living nematode Caenorhabditis elegans remains one of the most robust and flexible genetic systems for interrogating the complexities of animal biology. Targeted genetic manipulations, such as RNA interference (RNAi), CRISPR/Cas9- or array-based transgenesis, all depend on initial delivery of nucleic acids. Delivery of dsRNA by feeding can be effective, but the expression in Escherichia coli is not conducive to experiments intended to remain sterile or with defined microbial communities. Soaking-based delivery requires prolonged exposure of animals to high-material concentrations without a food source and is of limited throughput. Last, microinjection of individual animals can precisely deliver materials to animals’ germlines, but is limited by the need to target and inject each animal one-by-one. Thus, we sought to address some of these challenges in nucleic acid delivery by developing a population-scale delivery method. We demonstrate efficient electroporation-mediated delivery of dsRNA throughout the worm and effective RNAi-based silencing, including in the germline. Finally, we show that guide RNA delivered by electroporation can be utilized by transgenic Cas9 expressing worms for population-scale genetic targeting. Together, these methods expand the scale and scope of genetic methodologies that can be applied to the C. elegans system.

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Journal articles on the topic 'Soaking RNAi'

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