Cell-based assays

Regardless of the technology (RNAi, CRISPR, over-expression, etc.), a good cell-based assay is the best foundation for a cell-based screen. We have equipment, provide reagents, share protocols, and more to support development of high-throughput screen assays in Drosophila cells.

Reagents, consultation, and other support is available for screens off-site. We also support screens on-site at our facility. Assays can be done using a number of types of reagents, including reagents for knockdown or over-expression of protein-coding genes, and interrogation of miRNAs.

See links below to relevant reagents, protocols, publications, and more.

News

Cartoon of fly host cells with virus or endosymbiotic bacteria

Cell-based RNAi screening helps reveal host-microbe interactions--two new screen reports

November 19, 2018

Laboratories at the Skirball Institute at New York University and the Boyce Thompson Institute at Cornell University reported results of two different cell-based Drosophila RNAi screens in papers published this week. The screens have in common that they looked at interactions between the host insect cells and a microbe -- the endosymbiont Wolbachia in one study and baculovirus in the other. For more, check out the newly published studies. For both these screens, the DRSC provided libraries for screens that were then performed at the host institution.

 

... Read more about Cell-based RNAi screening helps reveal host-microbe interactions--two new screen reports
flySAM

Missed us at ADRC 2018? View our workshop slides!

April 19, 2018
Thank you to all those who attended our workshop at last week's Annual Drosophila Research Conference in Philadelphia, PA, USA. It was great to talk fly stocks, cell screens, and bioinformatics with the community. We are here to help and look forward to continued feedback on the resources we are building to empower your research. PDFs of our workshop presentations are attached to this news item. The slides will help you learn more about our in vivo resources for CRISPR, new pooled cell-based CRISPR screen technology, and bioinformatics resources at our facility.  Feel free to contact... Read more about Missed us at ADRC 2018? View our workshop slides!
Cartoon of essential gene pooled screen (made using BioRender.io)

Pooled-format CRISPR screens in Drosophila cells

March 22, 2018

The DRSC/TRiP-FGR is pleased to support collaborations on pooled CRISPR screens using the method recently, reported in eLife by Viswanatha et al. (PDF download file below).

From the abstract: "... Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness...

Read more about Pooled-format CRISPR screens in Drosophila cells
Photo of 384-well assay plates

Congrats to Sung, Shears, and colleagues: "Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress"

December 13, 2017

Eui Jae Sung, Stephen Shears, and colleagues have published a research report that includes a screen of dsRNAs from the DRSC reagent collection using S2 cells. We shipped dsRNA reagents to the lab for a screen at their home institution, in addition to providing consultation and data management support. The resulting study by Sung et al. was published on Dec. 11, 2017: Sung EJ, Ryuda M, Matsumoto H, Uryu O, Ochiai M, Cook ME, Yi NY, Wang H, Putney JW, Bird GS, Shears SB, Hayakawa Y. Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress...

Read more about Congrats to Sung, Shears, and colleagues: "Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress"
Figure 2 from Housden et al 2017 PNAS

Variable Dose Analysis: a new DRSC-supported cell screen approach that leverages existing reagents to perform robust screens

December 1, 2017

We are excited to report the publication of a paper from Benjamin Housden and colleagues describing development and use of the Variable Dose Analysis (VDA) approach. Ben developed a way to use existing TRiP shRNA plasmids originally developed for fly stock production in a new, effective approach to high-throughput cell screening.

The VDA approach is particularly useful for combinatorial approaches that are acutely sensitive to assay robustness. The screen Ben and colleagues report focused on synthetic effects in Drosophila tumor model cells.  The...

Read more about Variable Dose Analysis: a new DRSC-supported cell screen approach that leverages existing reagents to perform robust screens
Screenshot of a 2015 Science paper from Payre and colleagues

Francois Payre's plenary talk at ADRC 2017 features results from DRSC cell-based screen

March 30, 2017

Those of us lucky enough to be at the Annual Drosophila Research Conference this morning saw a great talk by Francois Payre about regulation of Shavenbaby by small ORFs. A genome-wide cell-based screen done at the DRSC by Emilie Benrabah identified the mechanism of regulation. As this exemplifies, cell screens can help identify key pathways and factors that can then be followed up with in vivo studies.

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Publications

Iiro Taneli Helenius, Ryan J Haake, Yong-Jae Kwon, Jennifer A Hu, Thomas Krupinski, Marina S Casalino-Matsuda, Peter HS Sporn, Jacob I Sznajder, and Greg J Beitel. 2016. “Identification of Drosophila Zfh2 as a Mediator of Hypercapnic Immune Regulation by a Genome-Wide RNA Interference Screen.” J Immunol, 196, 2, Pp. 655-67.Abstract

Hypercapnia, elevated partial pressure of CO2 in blood and tissue, develops in many patients with chronic severe obstructive pulmonary disease and other advanced lung disorders. Patients with advanced disease frequently develop bacterial lung infections, and hypercapnia is a risk factor for mortality in such individuals. We previously demonstrated that hypercapnia suppresses induction of NF-κB-regulated innate immune response genes required for host defense in human, mouse, and Drosophila cells, and it increases mortality from bacterial infections in both mice and Drosophila. However, the molecular mediators of hypercapnic immune suppression are undefined. In this study, we report a genome-wide RNA interference screen in Drosophila S2* cells stimulated with bacterial peptidoglycan. The screen identified 16 genes with human orthologs whose knockdown reduced hypercapnic suppression of the gene encoding the antimicrobial peptide Diptericin (Dipt), but did not increase Dipt mRNA levels in air. In vivo tests of one of the strongest screen hits, zinc finger homeodomain 2 (Zfh2; mammalian orthologs ZFHX3/ATBF1 and ZFHX4), demonstrate that reducing zfh2 function using a mutation or RNA interference improves survival of flies exposed to elevated CO2 and infected with Staphylococcus aureus. Tissue-specific knockdown of zfh2 in the fat body, the major immune and metabolic organ of the fly, mitigates hypercapnia-induced reductions in Dipt and other antimicrobial peptides and improves resistance of CO2-exposed flies to infection. Zfh2 mutations also partially rescue hypercapnia-induced delays in egg hatching, suggesting that Zfh2's role in mediating responses to hypercapnia extends beyond the immune system. Taken together, to our knowledge, these results identify Zfh2 as the first in vivo mediator of hypercapnic immune suppression.

Hirotaka Kanoh, Takayuki Kuraishi, Li-Li Tong, Ryo Watanabe, Shinji Nagata, and Shoichiro Kurata. 2015. “Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract.” Biochem Biophys Res Commun, 467, 2, Pp. 400-6.Abstract
Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that our larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.
Hirotaka Kanoh, Li-Li Tong, Takayuki Kuraishi, Yamato Suda, Yoshiki Momiuchi, Fumi Shishido, and Shoichiro Kurata. 2015. “Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults.” Sci Signal, 8, 400, Pp. ra107.Abstract
The Drosophila Toll pathway plays important roles in innate immune responses against Gram-positive bacteria and fungi. To identify previously uncharacterized components of this pathway, we performed comparative, ex vivo, genome-wide RNA interference screening. In four screens, we overexpressed the Toll adaptor protein dMyd88, the downstream kinase Pelle, or the nuclear factor κB (NF-κB) homolog Dif, or we knocked down Cactus, the Drosophila homolog of mammalian inhibitor of NF-κB. On the basis of these screens, we identified the E3 ubiquitin ligase Sherpa as being necessary for the activation of Toll signaling. A loss-of-function sherpa mutant fly exhibited compromised production of antimicrobial peptides and enhanced susceptibility to infection by Gram-positive bacteria. In cultured cells, Sherpa mediated ubiquitylation of dMyd88 and Sherpa itself, and Sherpa and Drosophila SUMO (small ubiquitin-like modifier) were required for the proper membrane localization of an adaptor complex containing dMyd88. These findings highlight a role for Sherpa in Drosophila host defense and suggest the SUMOylation-mediated regulation of dMyd88 functions in Toll innate immune signaling.
J Zanet, E Benrabah, T Li, A Pélissier-Monier, H Chanut-Delalande, B Ronsin, HJ Bellen, F Payre, and S Plaza. 2015. “Pri sORF peptides induce selective proteasome-mediated protein processing.” Science, 349, 6254, Pp. 1356-8.Abstract

A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

Stephanie E Mohr, Yanhui Hu, Kirstin Rudd, Michael Buckner, Quentin Gilly, Blake Foster, Katarzyna Sierzputowska, Aram Comjean, Bing Ye, and Norbert Perrimon. 2015. “Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells.” G3 (Bethesda), 5, 9, Pp. 1919-24.Abstract

RNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online.

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