Van den Brande S, Gijbels M, Wynant N, Santos D, Mingels L, Gansemans Y, Van Nieuwerburgh F, Vanden Broeck J. The presence of extracellular microRNAs in the media of cultured Drosophila cells. Sci Rep. 2018 Nov 23;8(1):17312. PMID: 30470777.
Abstract: "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."
Monday, November 26, 2018
Monday, November 19, 2018
RNAi screen used to identify host cell factors required for baculovirus entry
Hodgson JJ, Buchon N, Blissard GW. Identification of insect genes involved in baculovirus AcMNPV entry into insect cells. Virology. 2018 Nov 13;527:1-11. doi: 10.1016/j.virol.2018.10.022. PMID: 30445201.
Abstract: "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. "
Abstract: "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. "
Genome-wide screen for factors related to Wolbachia interaction with dipteran host cells
Grobler Y, Yun CY, Kahler DJ, Bergman CM, Lee H, Oliver B, Lehmann R. Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation. PLoS Pathog. 2018 Nov 13;14(11):e1007445. doi: 10.1371/journal.ppat.1007445. PMID: 30422992.
From the abstract: "Wolbachia 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."
From the abstract: "Wolbachia 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."
Thursday, November 15, 2018
Thinking about size: in vivo RNAi screen for neuronal genes related to energy homeostasis
Trinh I, Gluscencova OB, Boulianne GL. An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion. Mol Metab. 2018 Oct 19. pii: S2212-8778(18)30828-7. doi:10.1016/j.molmet.2018.10.006. PMID: 30389349.
From the abstract: "... Here, we have used the genetically tractable fruit fly, Drosophila melanogaster, 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, Diacylglycerol kinase (Dgk), 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."
From the abstract: "... Here, we have used the genetically tractable fruit fly, Drosophila melanogaster, 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, Diacylglycerol kinase (Dgk), 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."
Where am I? in vivo RNAi screen looks at larval chordotonal organs
Hassan A, Timerman Y, Hamdan R, Sela N, Avetisyan A, Halachmi N, Salzberg A. An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs. G3 (Bethesda). 2018 May 31;8(6):1871-1884. doi: 10.1534/g3.118.200218. PubMed PMID: 29678948; PubMed Central PMCID: PMC5982817.
From the abstract: "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."
From the abstract: "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."
Thursday, October 11, 2018
Viral protein blocks RNAi
Today in Cell Host & Microbe: Nayak et al. A Viral Protein Restricts Drosophila RNAi immunity by Regulating Argonaute Activity and Stability.
View the paper here.
View the paper here.
Wednesday, September 5, 2018
Video report -- cell-based assay
Peters KA, Detmar E, Sepulveda L, Del Valle C, Valsquier R, Ritz A, Rogers SL, Applewhite DA. A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells. J Vis Exp. 2018 Aug 19;(138). doi: 10.3791/58325. PubMed PMID: 30176023.
Monday, July 23, 2018
Engineered virus developed for RNAi reagent delivery
Taning CNT, Christiaens O, Li X, Swevers L, Casteels H, Maes M, Smagghe G. Engineered Flock House Virus for Targeted Gene Suppression Through RNAi in Fruit Flies (Drosophila melanogaster) in Vitro and in Vivo. Front Physiol. 2018 Jul 3;9:805. PMID: 30018564; PMCID: PMC6037854.
From the abstract: "... 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 Drosophila melanogaster embryos, and with adult stages of D. melanogaster. 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."
From the abstract: "... 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 Drosophila melanogaster embryos, and with adult stages of D. melanogaster. 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."
Monday, June 4, 2018
in vivo RNAi screen focused on the female germline
Cho Y, Lai CM, Lin KY, Hsu HJ. A Targeted RNAi Screen Reveals Drosophila Female-Sterile Genes That Control the Size of Germline Stem Cell Niche During Development. G3 (Bethesda). 2018 May 15. PMID: 29764959.
From the abstract: "... 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. ..."
From the abstract: "... 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. ..."
Genome-wide cell-based RNAi screen related to JAK/STAT signaling
Fisher KH, Fragiadaki M, Pugazhendhi D, Bausek N, Arredondo MA, Thomas SJ, Brown S, Zeidler MP. A genome-wide RNAi screen identifies MASK as a positive regulator of cytokine receptor stability. J Cell Sci. 2018 May 30. PMID: 29848658.
From the abstract: "... 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."
From the abstract: "... 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."
Thursday, May 31, 2018
New in vivo RNAi screen report -- larval chordotonal organ morphogenesis
An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs
Abeer Hassan, Yael Timerman, Rana Hamdan, Nitzan Sela, Adel Avetisyan, Naomi Halachmi and Adi Salzberg
G3: GENES, GENOMES, GENETICS June 1, 2018 vol. 8 no. 6 1871-1884
https://doi.org/10.1534/g3.118.200218
From the abstract: "... 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. ..."
Abeer Hassan, Yael Timerman, Rana Hamdan, Nitzan Sela, Adel Avetisyan, Naomi Halachmi and Adi Salzberg
G3: GENES, GENOMES, GENETICS June 1, 2018 vol. 8 no. 6 1871-1884
https://doi.org/10.1534/g3.118.200218
From the abstract: "... 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. ..."
in vivo fly RNAi screen identifies ortholog of SPO11 DNA break protein as pro-tumor in a fly tumor model
Rossi F, Molnar C, Hashiyama K, Heinen JP, Pampalona J, Llamazares S, Reina J, Hashiyama T, Rai M, Pollarolo G, Fernández-Hernández I, Gonzalez C. 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. Open Biol. 2017 Aug;7(8). pii: 170156. PMID: 28855394; PMCID: PMC5577452.
The abstract: "Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo. We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt 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 meiotic W68 (mei-W68), the Drosophila 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 Drosophila mei-W68/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 Drosophila."
The abstract: "Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo. We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt 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 meiotic W68 (mei-W68), the Drosophila 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 Drosophila mei-W68/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 Drosophila."
Tuesday, May 8, 2018
Drosophila cell-based RNAi screen related to treatment of epilepsy
Lin WH, He M, Fan YN, Baines RA. An RNAi-mediated screen identifies novel
targets for next-generation antiepileptic drugs based on increased expression of
the homeostatic regulator pumilio. J Neurogenet. 2018 May 2:1-12. PMID: 29718742.
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 Drosophila. 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, para[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."
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 Drosophila. 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, para[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."
Tuesday, March 6, 2018
FlyBook review on RNAi screening in Drosophila cells and in vivo
Heigwer F, Port F, Boutros M. RNA Interference (RNAi) Screening in Drosophila. Genetics. 2018 Mar;208(3):853-874. PMID: 29487145.
From the abstract: "... RNA interference (RNAi) ... has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, 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 Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. ..."
From the abstract: "... RNA interference (RNAi) ... has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, 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 Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. ..."
Friday, February 23, 2018
Fly iPCs? Opinion piece in Genes suggests it should be tried
Kaur P, Jin HJ, Lusk JB, Tolwinski NS. Modeling the Role of Wnt Signaling in Human and Drosophila Stem Cells. Genes (Basel). 2018 Feb 16;9(2). pii: E101. PMID: 29462894.
From the abstract: "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."
Of related interest: RasV12 method for isolation of new cell lines, developed by A. Simcox (OSU).
http://flyrnai.blogspot.com/2012/10/breaking-report-cell-culture.html
http://flyrnai.blogspot.com/2009/01/technical-note-making-new-cell-lines.html
From the abstract: "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."
Of related interest: RasV12 method for isolation of new cell lines, developed by A. Simcox (OSU).
http://flyrnai.blogspot.com/2012/10/breaking-report-cell-culture.html
http://flyrnai.blogspot.com/2009/01/technical-note-making-new-cell-lines.html
Wednesday, January 3, 2018
Single-molecule imaging study of a Wnt ligand using S2 and S2R+ Drosophila cultured cells
Lippert A, Janeczek AA, Fürstenberg A, Ponjavic A, Moerner WE, Nusse R, Helms JA, Evans ND, Lee SF. Single-Molecule Imaging of Wnt3A Protein Diffusion on Living Cell Membranes. Biophys J. 2017 Dec 19;113(12):2762-2767. PMID: 29262368.
Abstract: "Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport."
Abstract: "Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport."
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