Report of detection of miRNAs in cell-free media from cultured Drosophila cells

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."

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."

Methods publications relevant to cell and in vivo RNAi

Methods in Molecular Biology has recently published papers relevant to Drosophila cell culture, cell-based RNAi, and in vivo RNAi.

Debec A, Megraw TL, Guichet A. Methods to Establish Drosophila Cell Lines. Methods Mol Biol. 2016;1478:333-351. PubMed PMID: 27730593. 

Billmann M, Boutros M. Methods for High-Throughput RNAi Screening in Drosophila Cells. Methods Mol Biol. 2016;1478:95-116. PubMed PMID: 27730577.

Kaya-Çopur A, Schnorrer F. A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila. Methods Mol Biol. 2016;1478:117-143. PubMed PMID: 27730578.

Cultured Drosophila cells 'humanized' for drug studies--video methods paper

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5. https://t.co/Cykmii1hlq

— flypapers (@fly_papers) February 11, 2016

Drosophila cell team at the DGRC reports integrase-mediated cassette exchange approach to production of clonal transgenic fly cell lines

Lucy Cherbas1, Jennifer Hackney, Lei Gong, Claire Salzer, Eric Mauser, Dayu Zhang and Peter Cherbas. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines.
Early online in G3.

From the abstract: "We describe an adaptation of ΦC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking site lines carries a single mapped copy of one of the docking platforms. ... We describe procedures for isolating cells carrying the substitutions ... When compared with clonal lines made by traditional transformation methods ... targeted insertion lines give more uniform expression, lower basal expression and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays – a major emphasis of cell-based studies." 

DGRC team reports on targeted insertion approach to modifying Drosophila cultured cell lines

Lucy Cherbas, Jennifer Hackney, Lei Gong, Claire Salzer, Eric Mauser, Dayu Zhang and Peter Cherbas. 2015. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines. Early online at Genetics.

From the abstract: "We describe an adaptation of φC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. ... We demonstrated the technology by integrating a cassette containing a Cu⁺⁺-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays – a major emphasis of cell-based studies."

New at the DRSC website--protocols for single-cell cloning and stable transfection of Drosophila cells

Two new step-by-step protocols available at the DRSC website:

• Single-cell cloning of Drosophila cells
• Making stably transfected Drosophila cells

And check out these related publications and resources:

Housden BE, Lin S, Perrimon N. Cas9-based genome editing in Drosophila. Methods Enzymol. 2014;546:415-39. PMID: 25398351.

Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C,
Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. PMID: 26350902.

Santos MG, Jorge SA, Brillet K, Pereira CA. Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression. Cytotechnology. 2007 May;54(1):15-24. PMID: 19003014; PMCID: PMC2267513.

Have a qPCR machine but no software for high-resolution melt analysis following CRISPR modification? Check out the HRMA online tool.

Caffeine, kinases and the Hippo pathway: cell-based fly RNAi screen with in vivo follow-up

Di Cara F, Maile TM, Parsons BD, Magico A, Basu S, Tapon N, King-Jones K. The Hippo pathway promotes cell survival in response to chemical stress. Cell Death Differ. 2015 Mar 13. PMID: 26021298.

From the abstract: "Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. Stress signaling pathways activate multiple cellular programs that range from the activation of survival pathways to the initiation of cell death when cells are damaged beyond repair. To identify novel players acting in stress response pathways, we conducted a cell culture RNA interference (RNAi) screen using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, we examined how caffeine affects cell survival when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, we identified and validated 10 kinases and 4 phosphatases that are essential for cell survival under caffeine-induced stress both in cell culture and living flies. Remarkably, our screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. ... Our in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress."

Review and detailed protocols--CRISPR-Cas9 in flies and fly cells

Housden BE, Lin S, Perrimon N. Cas9-based genome editing in Drosophila. Methods Enzymol. 2014;546:415-39. PMID: 25398351.  

From the abstract: "... we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals."

Includes helpful tables listing sgRNA design tools, relevant fly stocks and plasmids.

Cell-based assays--new reports

Ramdas NM, Shivashankar GV. Cytoskeletal Control of Nuclear Morphology and Chromatin Organization. J Mol Biol. 2014 Oct 2. pii: S0022-2836(14)00495-1. PMID: 25281900.

From the abstract: "... We demonstrate here the differential influence of perinuclear actin- and microtubule-driven assemblies on nuclear architecture using pharmacological inhibitors and targeted RNA interference knockdown of cytoskeleton components in Drosophila cells. We find evidence that the loss of perinuclear actin assembly results in basolateral enhancement of microtubule organization and this is reflected functionally by enhanced nuclear dynamics. ..."

Ribeiro SA, D'Ambrosio MV, Vale RD. Induction of Focal Adhesions and Motility in Drosophila S2 cells. Mol Biol Cell. 2014 PMID: 25273555.

From the abstract: "... Here, we describe a system for inducing the formation of focal adhesions in normally non-ECM-adherent, non-motile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a non-polarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. ... These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility."

Full-genome screen related to functional insulators

Another full-genome DRSC screen published--congrats!

Fig. 1 from the Bohla et al. paper.

Bohla D, Herold M, Panzer I, Buxa MK, Ali T, Demmers J, Krüger M, Scharfe M, Jarek M, Bartkuhn M, Renkawitz R. A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking. PLoS One. 2014 Sep 23;9(9):e107765. PubMed PMID: 25247414; PubMed Central PMCID: PMC4172637.

From the abstract: "... Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. ..."

Study related to Abl kinase uses primary cultured cells

This is not an RNAi study but the approach taken might of interest to those thinking about cell-based studies. Liu L, Wu CF. Distinct effects of Abelson kinase mutations on myocytes and neurons in dissociated Drosophila embryonic cultures: mimicking of high temperature. PLoS One. 2014 Jan 21;9(1):e86438. PMID: 24466097; PMCID: PMC3897706. From the abstract: "... we observed myocyte fusion events and nerve-muscle contact formation between WT and Abl cells in mixed WT and Abl cultures derived from labeled embryos ..."

Study explores culture media for Drosophila cells

Burnette M, Brito-Robinson T, Li J, Zartman J. An inverse small molecule screen to design a chemically defined medium supporting long-term growth of Drosophila cell lines. Mol Biosyst. 2014 Aug 6. PMID: 25096480.

From the abstract: "Drosophila cell culture is used as a model system with multiple applications ...  To characterize the minimal requirements for long-term maintenance of Drosophila cell lines, we developed an inverse screening strategy to identify small molecules and synergies stimulating proliferation in a chemically defined medium. ... Validated factors were investigated for their ability to maintain cell growth over multiple passages in the chemically defined medium (CDM). The polyamine spermidine proved to be the critical component that enables the CDM to support long-term maintenance of Cl.8 cells. Spermidine supplementation upregulates DNA synthesis for Cl.8 and S2 cells and increases MAPK signaling for Cl.8 cells. The CDM also supports the long-term growth of Kc167 cells. Our target scoring approach validated the importance of polyamines ... Future iterations of the screen will enable the identification of compound combinations optimized for specific applications ... thus increasing the versatility of Drosophila cell culture as both a genetic and biochemical model system. ..."

Cell-based assays related to Notch signaling

Xu A, Irvine KD. Notch-ligand binding assays in Drosophila cells. Methods Mol Biol. 2014;1187:277-84. PMID: 25053497.

 Li J, Housden BE, Bray SJ. Notch signaling assays in Drosophila cultured cell lines. Methods Mol Biol. 2014;1187:131-41. PMID: 25053486.

Luciferase-based assays in cultured cells

Yun C, Dasgupta R. Luciferase reporter assay in Drosophila and Mammalian tissue culture cells. Curr Protoc Chem Biol. 2014 Mar 14;6(1):7-23. PMID: 24652620.

Use of fly cells to study the cytoskeleton

Nye J, Buster DW, Rogers GC. The use of cultured Drosophila cells for studying the microtubule cytoskeleton. Methods Mol Biol. 2014;1136:81-101. PMID: 24633795.

From the abstract:  The authors "describe basic techniques for gene knockdown, transgene expression, preparation for fluorescence microscopy, and centrosome enrichment using cultured Drosophila cells with an emphasis on studying the microtubule cytoskeleton."

Life in a bubble

At the DRSC we recently tested a new commercial plate type supporting growth of cells in very small volumes of media--tiny domes of liquid in 96- or 384-well format. We have not had time for much testing but we can say this:  S2R+ cultured Drosophila cells grew just fine in this format in our hands. Photo below. Feel free to email the DRSC Director if you have any questions. DropArray plates from Curiox. Posting this for information purposes only (should not be considered an endorsement).

Methods paper on fly cell lines

This new paper in Methods from L. Cherbas & L. Gong should be a great resource for those interested in using Drosophila cells in research studies.

Cherbas L, Gong L. Cell lines. Methods. 2014 Jan 13. PMID: 24434506.

CRISPR/Cas9 in Drosophila cultured cells

RNAi screening in customized Drosophila cell lines has seemed like an inevitability since the emergence of the new genome engineering approaches.  With this report, that eventuality seems another step closer.

Bassett AR, Tibbit C, Ponting CP, Liu JL. Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9. Biol Open. 2013 Dec 10. PMID: 24326186.

Fly RNAi and Legionella. Recent book chapter.

De Jesús DA, O'Connor TJ, Isberg RR. Analysis of Legionella Infection Using RNAi in Drosophila Cells. Methods Mol Biol. 2013;954:251-64. doi: 10.1007/978-1-62703-161-5_15. PubMed PMID: 23150401.