tag:blogger.com,1999:blog-35004259350891438102024-03-13T21:59:38.927-04:00FlyRNAiHelpful information about cells, assays, RNAi and more, plus the occasional weird fly fact, brought to you by the Director of the Drosophila RNAi Screening Center (DRSC) at HMS.Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.comBlogger622125tag:blogger.com,1999:blog-3500425935089143810.post-40053513433773788322021-06-21T08:34:00.003-04:002021-06-21T08:34:12.368-04:00Of interst: Chen et al. "Transcript level is a key factor affecting RNAi efficiency"<p>Pestic Biochem Physiol. 2021 Jul;176:104872. doi: 10.1016/j.pestbp.2021.104872.</p><p><b>Transcript level is a key factor affecting RNAi efficiency.</b><br /><br />Chen J, Peng Y, Zhang H, Wang K, Tang Y, Gao J, Zhao C, Zhu G, Palli SR, Han Z<br /></p><p><b>Abstract:</b><br /></p><p>Efficiency is the basis for the application of RNA interference (RNAi) technology. Actually, RNAi efficiency varies greatly among insect species, tissues and genes. Previous efforts have revealed the mechanisms for variation among insect species and tissues. Here, we investigated the reason for variable efficiency among the target genes in the same insect. First, we tested the genes sampled randomly from <i>Tribolium castaneum, Locusta migratoria</i> and <i>Drosophila</i> S2 cells for both their expression levels and sensitivity to RNAi. The results indicated that the genes with higher expression levels were more sensitive to RNAi. Statistical analysis showed that the correlation coefficients between transcript levels and knockdown efficiencies were 0.8036 (n = 90), 0.7255 (n = 18) and 0.9505 (n = 13), respectively in <i>T. castaneum, L. migratoria</i> and <i>Drosophila</i> S2 cells. Subsequently, ten genes with varied expression level in <br />different tissues (midgut and carcass without midgut) of <i>T. castaneum</i> were tested. The results indicated that the higher knockdown efficiency was always obtained in the tissue where the target gene expressed higher. In addition, three genes were tested in different developmental stages, larvae and pupae of <i>T. castaneum</i>. The results found that when the expression level increased after insect pupation, these genes became more sensitive to RNAi. Thus, all the proofs support unanimously that transcript level is a key factor affecting RNAi sensitivity. This finding allows for a better understanding of the RNAi efficiency variation and lead to effective or efficient use of RNAi technology.<br /><br />DOI: 10.1016/j.pestbp.2021.104872<br />PMID: 34119217 [Indexed for MEDLINE]</p>Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-70152308694838664012019-09-30T09:58:00.003-04:002019-09-30T09:58:45.279-04:00Two new in vivo Drosophila RNAi screens reportedZhou J, Xu L, Duan X, Liu W, Zhao X, Wang X, Shang W, Fang X, Yang H, Jia L, Bai J, Zhao J, Wang L, Tong C. <b>Large-scale RNAi screen identified Dhpr as a regulator of mitochondrial morphology and tissue homeostasis.</b> Sci Adv. 2019 Sep 18;5(9):eaax0365. PubMed PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/31555733">31555733</a>; PubMed Central PMCID: PMC6750926.<br />
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Umer Z, Akhtar J, Khan MHF, Shaheen N, Haseeb MA, Mazhar K, Mithani A, Anwar S, Tariq M. <b>Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator. </b>Epigenetics Chromatin. 2019 Sep 23;12(1):55. PubMed PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/31547845">31547845</a>.Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-59098874622925147372019-09-04T10:28:00.001-04:002019-09-04T10:28:27.604-04:00Drosophila Models of Human Disease: Whole-animal fly system facilitates study relevant...<a href="https://flydiseasemodels.blogspot.com/2019/09/whole-animal-fly-system-facilitates.html?spref=bl">Drosophila Models of Human Disease: Whole-animal fly system facilitates study relevant...</a>: Mishra-Gorur K, Li D, Ma X, Yarman Y, Xue L, Xu T. Spz/Toll-6 signal guides organotropic metastasis in Drosophila. Dis Model Mech. 2019 Sep...Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-54506240559991047252019-08-19T10:42:00.000-04:002019-08-19T10:42:10.909-04:00New S2 cell assay reported -- Cell densityRomine ML, Li M, Liu KJ, Patel SK, Nelson JG, Shen P, Cai HN. <b>A Cell Density-Dependent Reporter in the Drosophila S2 Cells.</b> Sci Rep. 2019 Aug 14;9(1):11868. doi: 10.1038/s41598-019-47652-0. PubMed PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/31413273">31413273</a>.
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<b><i>Abstract</i></b>: "Cell density regulates many aspects of cell properties and behaviors including metabolism, growth, cytoskeletal structure and locomotion. Importantly, the responses by cultured cells to density signals also uncover key mechanisms that govern animal development and diseases in vivo. Here we characterized a density-responsive reporter system in transgenic Drosophila S2 cells. We show that the reporter genes are strongly induced in a cell density-dependent and reporter-independent fashion. The rapid and reversible induction occurs at the level of mRNA accumulation. We show that multiple DNA elements within the transgene sequences, including a metal response element from the metallothionein gene, contribute to the reporter induction. The reporter induction correlates with changes in multiple cell density and growth regulatory pathways including hypoxia, apoptosis, cell cycle and cytoskeletal organization. Potential applications of such a density-responsive reporter will be discussed."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-69623405524691706302019-08-12T11:32:00.002-04:002019-08-12T11:32:48.283-04:00in vivo RNAi screen in the wingRotelli MD, Bolling AM, Killion AW, Weinberg AJ, Dixon MJ, Calvi BR. <b>An RNAi Screen for Genes Required for Growth of Drosophila Wing Tissue.</b> G3 (Bethesda). 2019 Aug 6. pii: g3.400581.2019. doi: 10.1534/g3.119.400581. PubMed PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/31387856">31387856</a>.<br />
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<b>Abstract:<i></i></b> "Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila. The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or βNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development." Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-34733053740562117132019-04-30T10:15:00.001-04:002019-04-30T10:15:05.720-04:00in vivo RNAi screen for metabolic enzymes required for eye developmentRose C. Pletcher, Sara L. Hardman, Sydney F. Intagliata, Rachel L. Lawson, Aumunique Page and Jason M. Tennessen<br />
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<b>A Genetic Screen Using the <i>Drosophila melanogaster</i> TRiP RNAi Collection To Identify Metabolic Enzymes Required for Eye Development</b><br />
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G3: GENES, GENOMES, GENETICS Early online April 29, 2019; https://doi.org/10.1534/g3.119.400193<br />
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<a href="http://www.g3journal.org/content/early/2019/04/29/g3.119.400193">http://www.g3journal.org/content/early/2019/04/29/g3.119.400193</a><br />
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<i>Abstract</i>: "The metabolic enzymes that compose glycolysis, the citric acid cycle, and other pathways within central carbon metabolism have emerged as key regulators of animal development. These enzymes not only generate the energy and biosynthetic precursors required to support cell proliferation and differentiation, but also moonlight as regulators of transcription, translation, and signal transduction. Many of the genes associated with animal metabolism, however, have never been analyzed in a developmental context, thus highlighting how little is known about the intersection of metabolism and development. Here we address this deficiency by using the Drosophila TRiP RNAi collection to disrupt the expression of over 1,100 metabolism-associated genes within cells of the eye imaginal disc. Our screen not only confirmed previous observations that oxidative phosphorylation serves a critical role in the developing eye, but also implicated a host of other metabolic enzymes in the growth and differentiation of this organ. Notably, our analysis revealed a requirement for glutamine and glutamate metabolic processes in eye development, thereby revealing a role of these amino acids in promoting Drosophila tissue growth. Overall, our analysis highlights how the Drosophila eye can serve as a powerful tool for dissecting the relationship between development and metabolism."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-88029321423044587292019-04-30T10:11:00.002-04:002019-04-30T10:11:53.104-04:00RNAi screen for genes in escort cells related to germ cell maintenance and differentiationGao Y, Mao Y, Xu RG, Zhu R, Zhang M, Sun J, Shen D, Peng P, Xie T, Ni JQ. <b>Defining gene networks controlling the maintenance and function of the differentiation niche by an in vivo systematic RNAi screen.</b> J Genet Genomics. 2019 Jan 20;46(1):19-30. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30745214">30745214</a>.<br />
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<i>Abstract</i>: "In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. In this study, we identified 173 EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-50488053956741975012019-04-30T10:09:00.001-04:002019-04-30T10:09:23.584-04:00Optogenetic tools for Drosophila S2 cellsOsswald M, Santos AF, Morais-de-Sá E. <b>Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells.</b> Biomolecules. 2019 Feb 12;9(2). pii: E61. doi: 10.3390/biom9020061. PubMed PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30759894">30759894</a>; PubMed Central PMCID: PMC6406598.<br />
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<i>Abstract</i>: "Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-16352948764900751582019-02-11T12:19:00.004-05:002019-02-11T12:19:55.433-05:00Screen explores possible roles of ion channels in Drosophila wing developmentGeorge LF, Pradhan SJ, Mitchell D, Josey M, Casey J, Belus MT, Dahal GR, Bates EA. <b>Ion Channel Contributions to Wing Development in <i>Drosophila melanogaster</i>.</b> G3 (Bethesda). 2019 Feb 7. pii: g3.400028.2019. doi: 10.1534/g3.119.400028. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30733380">30733380</a>.<br />
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<i>Abstract:</i> "During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The <i>Drosophila melanogaster </i>wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. We conducted a screen of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. Here we identify 44 candidates that significantly impacted development of the <i>Drosophila melanogaster</i> wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in our screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of our screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in our screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. Our results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-46286487688663805442019-02-11T12:17:00.000-05:002019-02-11T12:17:51.317-05:00Large-scale RNAi screen in red flour beetle (Tribolium) to identify genes involved in muscle developmentSchultheis D, Weißkopf M, Schaub C, Ansari S, Dao VA, Grossmann D, Majumdar U, Hakeemi MS, Troelenberg N, Richter T, Schmitt-Engel C, Schwirz J, Ströhlein N, Teuscher M, Bucher G, Frasch M. <b>A Large Scale Systemic RNAi Screen in the Red Flour Beetle Tribolium castaneum Identifies Novel Genes Involved in Insect Muscle Development.</b> G3 (Bethesda). 2019 Feb 7. pii: g3.200995.2018. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30733381">30733381</a>.<br />
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Schultheis D, Schwirz J, Frasch M. <b>RNAi Screen in Tribolium Reveals
Involvement of F-BAR Proteins in Myoblast Fusion and Visceral Muscle
Morphogenesis in Insects. </b>G3 (Bethesda). 2019 Feb 7. pii: g3.200996.2018. doi:
10.1534/g3.118.200996. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30733382">30733382</a>.Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-23343906376975129802018-11-26T12:04:00.001-05:002018-11-26T12:04:56.923-05:00Report of detection of miRNAs in cell-free media from cultured Drosophila cellsVan den Brande S, Gijbels M, Wynant N, Santos D, Mingels L, Gansemans Y, Van Nieuwerburgh F, Vanden Broeck J. <b>The presence of extracellular microRNAs in the media of cultured Drosophila cells.</b> Sci Rep. 2018 Nov 23;8(1):17312. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30470777">30470777</a>.
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<br />
<i>Abstract</i>: "While regulatory RNA pathways, such as RNAi, have commonly been described at an intracellular level, studies investigating extracellular RNA species in insects are lacking. In the present study, we demonstrate the presence of extracellular microRNAs (miRNAs) in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), we analysed the presence of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates, obtained from the cell-free conditioned media of S2 and Cl.8 cell cultures. Next-generation RNA-sequencing data confirmed our qRT-PCR results and provided evidence for selective miRNA secretion in EVs. To our knowledge, this is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects, the most speciose group of animals."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-87470663137803906962018-11-19T11:37:00.002-05:002018-11-19T11:37:33.574-05:00RNAi screen used to identify host cell factors required for baculovirus entryHodgson JJ, Buchon N, Blissard GW. <b>Identification of insect genes involved in baculovirus AcMNPV entry into insect cells.</b> Virology. 2018 Nov 13;527:1-11. doi: 10.1016/j.virol.2018.10.022. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30445201">30445201</a>.<br />
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<i>Abstract:</i> "The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a model enveloped DNA virus that infects and replicates in lepidopteran insect cells, and can efficiently enter a wide variety of non-host cells. Budded virions of AcMNPV enter cells by endocytosis and traffic to the nucleus where the virus initiates gene expression and genome replication. While trafficking of nucleocapsids by actin propulsion has been studied in detail, other important components of trafficking during entry remain poorly understood. We used a recombinant AcMNPV virus expressing an EGFP reporter in combination with an RNAi screen in Drosophila DL1 cells, to identify host proteins involved in AcMNPV entry. The RNAi screen targeted 86 genes involved in vesicular trafficking, including genes coding for VPS and ESCRT proteins, Rab GTPases, Exocyst proteins, and Clathrin adaptor proteins. We identified 24 genes required for efficient virus entry and reporter expression, and 4 genes that appear to restrict virus entry.
"Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-32265900100686329872018-11-19T11:35:00.002-05:002018-11-19T11:37:44.871-05:00Genome-wide screen for factors related to Wolbachia interaction with dipteran host cellsGrobler Y, Yun CY, Kahler DJ, Bergman CM, Lee H, Oliver B, Lehmann R. <b>Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation.</b> PLoS Pathog. 2018 Nov 13;14(11):e1007445. doi: 10.1371/journal.ppat.1007445. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30422992">30422992</a>.<br />
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<i>From the abstract: </i>"<i>Wolbachia</i> is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. ... We coupled genome-wide RNAi screening with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. ... This work provides evidence for Wolbachia-host translation interaction and strengthens our general understanding of the Wolbachia-host intracellular relationship."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-87055214143210021392018-11-15T12:50:00.003-05:002018-11-15T12:50:56.022-05:00Thinking about size: in vivo RNAi screen for neuronal genes related to energy homeostasisTrinh I, Gluscencova OB, Boulianne GL. <b>An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion. </b>Mol Metab. 2018 Oct 19. pii: S2212-8778(18)30828-7. doi:10.1016/j.molmet.2018.10.006. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30389349">30389349</a>.<br />
<br />
<i>From the abstract</i>: "... Here, we have used the genetically tractable fruit fly, <i>Drosophila melanogaster</i>, to identify genes/pathways that function in the nervous system to regulate energy balance.
... We performed an in vivo RNAi screen in Drosophila neurons and assayed for obese or lean phenotypes by measuring changes in levels of stored fats (in the form of triacylglycerides or TAG). Three rounds of screening were performed to verify the reproducibility and specificity of the adiposity phenotypes. Genes that produced >25% increase in TAG (206 in total) underwent a second round of screening to verify their effect on TAG levels by retesting the same RNAi line to validate the phenotype. All remaining hits were screened a third time by testing the TAG levels of additional RNAi lines against the genes of interest to rule out any off-target effects.
... We identified 24 genes including 20 genes that have not been previously associated with energy homeostasis. One identified hit, <i>Diacylglycerol kinase (Dgk)</i>, has mammalian homologues that have been implicated in genome-wide association studies for metabolic defects. ... Altogether, we have identified several genes that act within the CNS to regulate energy homeostasis. One of these, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-14351645598321925672018-11-15T12:45:00.001-05:002018-11-15T12:45:27.538-05:00Where am I? in vivo RNAi screen looks at larval chordotonal organsHassan A, Timerman Y, Hamdan R, Sela N, Avetisyan A, Halachmi N, Salzberg A. <b>An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs.</b> G3 (Bethesda). 2018 May 31;8(6):1871-1884. doi: 10.1534/g3.118.200218. PubMed PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/29678948">29678948</a>; PubMed Central PMCID: PMC5982817.<br />
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<i>From the abstract:</i> "The proprioceptive chordotonal organs (ChO) of a fly larva respond to mechanical stimuli generated by muscle contractions and consequent deformations of the cuticle. The ability of the ChO to sense the relative displacement of its epidermal attachment sites likely depends on the correct mechanical properties of the accessory (cap and ligament) and attachment cells that connect the sensory unit (neuron and scolopale cell) to the cuticle. ... Here we describe an RNAi screen that focused on the ChO's accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. Most interestingly, one phenotypic group consisted of genes that affected the response of specific ChO cell types to developmental organ stretching, leading to abnormal pattern of cell elongation. The 'cell elongation' group included the transcription factors Delilah and Stripe ... Other genes found to affect the pattern of ChO cell elongation ... represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-24581923263304995952018-10-11T11:55:00.003-04:002018-10-11T11:55:30.928-04:00Viral protein blocks RNAiToday in <i>Cell Host & Microbe</i>: Nayak et al. <b>A Viral Protein Restricts Drosophila RNAi immunity by Regulating Argonaute Activity and Stability. </b><br />
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View the paper <a href="https://www.cell.com/cell-host-microbe/fulltext/S1931-3128(18)30490-6">here</a>.<br />
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Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-74627374507693423802018-09-05T09:31:00.001-04:002018-09-05T09:31:22.121-04:00Video report -- cell-based assayPeters KA, Detmar E, Sepulveda L, Del Valle C, Valsquier R, Ritz A, Rogers SL, Applewhite DA. <b>A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells. </b>J Vis Exp. 2018 Aug 19;(138). doi: 10.3791/58325. PubMed PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30176023">30176023</a>.Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-67393694446267184022018-07-23T12:28:00.000-04:002018-07-23T12:28:06.659-04:00Engineered virus developed for RNAi reagent deliveryTaning CNT, Christiaens O, Li X, Swevers L, Casteels H, Maes M, Smagghe G. <b>Engineered Flock House Virus for Targeted Gene Suppression Through RNAi in Fruit Flies (<i>Drosophila melanogaster</i>) in Vitro and in Vivo. </b>Front Physiol. 2018 Jul 3;9:805. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/30018564">30018564</a>; PMCID: PMC6037854.<br />
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<i>From the abstract</i>: "... In this study, we investigated the ability of engineered Flock House virus (FHV) to induce targeted gene suppression through RNAi under in vitro and in vivo condition. As proxy for fruit flies of agricultural importance, we worked with S2 cells as derived from <i>Drosophila melanogaster</i> embryos, and with adult stages of <i>D. melanogaster</i>. We found that the expression level for all of the targeted genes were reduced by more than 70% in both the in vitro and in vivo bioassays. Furthermore, the cell viability and median survival time bioassays demonstrated that the recombinant FHV expressing target gene sequences caused a significantly higher mortality (60-73% and 100%) than the wild type virus (24 and 71%), in both S2 cells and adult insects, respectively. This is the first report showing that a single stranded RNA insect virus such as FHV, can be engineered as an effective in vitro and in vivo RNAi delivery system. Since FHV infects many insect species, the described method could be exploited to improve the efficiency of dsRNA delivery for RNAi-related studies in both FHV susceptible insect cell lines and live insects that are recalcitrant to the uptake of naked dsRNA."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-48067421022972639412018-06-04T10:06:00.001-04:002018-06-04T10:06:26.092-04:00in vivo RNAi screen focused on the female germlineCho Y, Lai CM, Lin KY, Hsu HJ. <b>A Targeted RNAi Screen Reveals Drosophila Female-Sterile Genes That Control the Size of Germline Stem Cell Niche During Development.</b> G3 (Bethesda). 2018 May 15. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/29764959">29764959</a>.<br />
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<i>From the abstract</i>: "... we conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. ..."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-64934545529183461912018-06-04T08:14:00.000-04:002018-06-04T08:14:31.015-04:00Genome-wide cell-based RNAi screen related to JAK/STAT signalingFisher KH, Fragiadaki M, Pugazhendhi D, Bausek N, Arredondo MA, Thomas SJ, Brown S, Zeidler MP. <b>A genome-wide RNAi screen identifies MASK as a positive regulator of cytokine receptor stability.</b> J Cell Sci. 2018 May 30. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/29848658">29848658</a>.<br />
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<i>From the abstract:</i> "... In order to transduce ligand activation, cytokine receptors must dimerise. However, mechanisms regulating their dimerisation are poorly understood. In order to better understand the processes regulating cytokine receptor levels, activity and dimerisation, we used the highly conserved JAK/STAT pathway in Drosophila, which acts via a single receptor, known as Domeless. We have performed a genome-wide RNAi screen in Drosophila cells, identifying MASK as a positive regulator of Domeless dimerisation and protein levels. ... our results identify MASK as a novel regulator of cytokine receptor levels, and suggest functional conservation, which may have implications for human health."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-60045054378914157422018-05-31T12:48:00.001-04:002018-05-31T12:48:42.052-04:00New in vivo RNAi screen report -- larval chordotonal organ morphogenesis<b>An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs</b><br />
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Abeer Hassan, Yael Timerman, Rana Hamdan, Nitzan Sela, Adel Avetisyan, Naomi Halachmi and Adi Salzberg<br />
G3: GENES, GENOMES, GENETICS June 1, 2018 vol. 8 no. 6 1871-1884<br />
<a href="https://doi.org/10.1534/g3.118.200218">https://doi.org/10.1534/g3.118.200218</a><br />
<br />
<i>From the abstract</i>: "... Here we describe an RNAi screen that focused on the ChO’s accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. ..."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-32601673938838218402018-05-31T08:14:00.002-04:002018-05-31T08:14:29.023-04:00in vivo fly RNAi screen identifies ortholog of SPO11 DNA break protein as pro-tumor in a fly tumor modelRossi F, Molnar C, Hashiyama K, Heinen JP, Pampalona J, Llamazares S, Reina J, Hashiyama T, Rai M, Pollarolo G, Fernández-Hernández I, Gonzalez C. <b>An in vivo genetic screen in Drosophila identifies the orthologue of human cancer/testis gene SPO11 among a network of targets to inhibit lethal(3)malignant brain tumour growth.</b> Open Biol. 2017 Aug;7(8). pii: 170156. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/28855394">28855394</a>; PMCID: PMC5577452.<br />
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<i>The abstract: </i>"Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of <i>lethal(3)malignant brain tumour</i> in <i>Drosophila in vivo</i>. We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in <i>mbt</i> tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is <i>meiotic W68</i> (<i>mei-W68</i>), the <i>Drosophila</i> orthologue of the human cancer/testis gene Sporulation-specific protein 11 (SPO11), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that <i>Drosophila</i> <i>mei-W68</i>/SPO11 drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a SPO11 orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like <i>Drosophila</i>."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-90654973211415974442018-05-08T09:12:00.000-04:002018-05-08T09:12:27.988-04:00Drosophila cell-based RNAi screen related to treatment of epilepsyLin WH, He M, Fan YN, Baines RA. <b>An RNAi-mediated screen identifies novel
targets for next-generation antiepileptic drugs based on increased expression of
the homeostatic regulator <i>pumilio</i></b>. <i>J Neurogenet</i>. 2018 May 2:1-12. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/29718742">29718742</a>.<br />
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From the abstract: "Despite availability of a diverse range of anti-epileptic drugs (AEDs), only
about two-thirds of epilepsy patients respond well to drug treatment. Thus, novel
targets are required to catalyse the design of next-generation AEDs. Manipulation
of neuron firing-rate homoeostasis, through enhancing Pumilio (Pum) activity, has
been shown to be potently anticonvulsant in <i>Drosophila</i>. In this study, we
performed a genome-wide RNAi screen in S2R + cells, using a luciferase-based dPum
activity reporter and identified 1166 genes involved in dPum regulation. Of these
genes, we focused on 699 genes that, on knock-down, potentiate dPum
activity/expression. Of this subgroup, 101 genes are activity-dependent based on
comparison with genes previously identified as activity-dependent by
RNA-sequencing. ... To test for anticonvulsant activity, we utilised an RNA-interference
approach in vivo. RNAi-mediated knockdown showed that 57/101 genes (61%) are
sufficient to significantly reduce seizure duration in the characterized seizure
mutant, <i>para</i>[bss]. ... this study validates Pum as a favourable target for
AED design and, moreover, identifies a number of lead compounds capable of
increasing the expression of this homeostatic regulator."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-17115181717409728012018-03-06T11:10:00.000-05:002018-03-06T11:10:16.111-05:00FlyBook review on RNAi screening in Drosophila cells and in vivoHeigwer F, Port F, Boutros M. <b>RNA Interference (RNAi) Screening in Drosophila.</b> Genetics. 2018 Mar;208(3):853-874. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/29487145">29487145</a>.<br />
<br />
<i>From the abstract:</i> "... RNA interference (RNAi) ... has had an important impact on identifying and characterizing gene function. First discovered in <i>Caenorhabditis elegans</i>, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In <i>Drosophila</i>, RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function in <i>Drosophila</i> with a particular focus on high-throughput screening methods applied in cultured cells. ..."Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0tag:blogger.com,1999:blog-3500425935089143810.post-67917410816191133862018-02-23T08:17:00.002-05:002018-02-23T08:32:46.509-05:00Fly iPCs? Opinion piece in Genes suggests it should be triedKaur P, Jin HJ, Lusk JB, Tolwinski NS. <b>Modeling the Role of Wnt Signaling in Human and Drosophila Stem Cells.</b> Genes (Basel). 2018 Feb 16;9(2). pii: E101. PMID: <a href="http://ncbi.nlm.nih.gov/pubmed/29462894">29462894</a>.<br />
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<i>From the abstract</i>: "The discovery of induced pluripotent stem (iPS) cells ... dramatically transformed the study of stem cells ... Although advances have pushed the field forward, human application remains some years away, in part due to the need for an in-depth mechanistic understanding. The role of Wnts in stem cells predates the discovery of iPS cells with Wnts established as major pluripotency promoting factors. Most work to date has been done using mouse and tissue culture models and few attempts have been made in other model organisms, but the recent combination of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing with iPS cell technology provides a perfect avenue for exploring iPS cells in model organisms. ... In this opinion article, we draw parallels between Wnt signaling in human and Drosophila stem cell systems, propose ways to obtain Drosophila iPS cells, and suggest ways to exploit the versatility of the Drosophila system for future stem cell studies."<br />
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<i>Of related interest</i>: RasV12 method for isolation of new cell lines, developed by A. Simcox (OSU).<br />
<a href="http://flyrnai.blogspot.com/2012/10/breaking-report-cell-culture.html">http://flyrnai.blogspot.com/2012/10/breaking-report-cell-culture.html</a><br />
<a href="http://flyrnai.blogspot.com/2009/01/technical-note-making-new-cell-lines.html">http://flyrnai.blogspot.com/2009/01/technical-note-making-new-cell-lines.html</a>Stephanie Mohrhttp://www.blogger.com/profile/07366059209046790720noreply@blogger.com0