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

Image of green fluorescence in GFP-tagged Drosophila cultured cells

New cell lines & new understandings using cutting-edge techniques

February 2, 2024

As a facility that supports large-scale screens in Drosophila and other insect cell lines, we get excited about reports of new Drosophila cell lines and related info.

We'd like to highlight two recent papers.

One report, a collaboration between Amanda Simcox's group, the DGRC, and our group here at the DRSC, describes new cell lines made in Amanda's group and characterized in a collaboration of the three groups. Muscle cells that pulse? Yes. That and other exciting new cell lines are reported in the publication below, and the cells are available at the DGRC...

Read more about New cell lines & new understandings using cutting-edge techniques
Decorative cartoon drawn with BioRender depicting DRSC-BTRR technology concepts

So you want to do a CRISPR pooled screen in insect cells? You can! Here's how

May 12, 2022

At the DRSC-BTRR, we've been doing a lot of pooled-format CRISPR knockout screens in Drosophila cells. We're finding the results to be robust and reproducible. And best of all, the results have been informative, providing insights into diverse areas of biology.

Thinking about how to do CRISPR knockout screens in cells is a little different from thinking about how to do a genetic or RNAi screen in vivo or doing an arrayed-format RNAi screen....

Read more about So you want to do a CRISPR pooled screen in insect cells? You can! Here's how
Graphical image of tissue culture, fly pushing, and computer, and the team of people who work with them

DRSC/TRiP and DRSC-BTRR Office Hours

September 13, 2021

New this fall: Online office hours!

Do you have questions about modifying Drosophila cell lines with CRISPR or performing large-scale cell screens? Questions about in vivo RNAi with TRiP fly stocks or CRISPR knockout or activation with our sgRNA fly stocks? Questions about our new protocols and resources for CRISPR mosquito cell lines? Pop into our Zoom office hours to say hello and get our expert input! Registration is required (see below).

DRSC/TRiP & DRSC-BTRR Office Hours Schedule:

Mon. Sept. 27, 2021, 12...

Read more about DRSC/TRiP and DRSC-BTRR Office Hours
Graphical image of tissue culture, fly pushing, and computer, and the team of people who work with them

DRSC-Biomedical Technology Research Resource

October 21, 2019

We are pleased to announce that we have been funded by NIH NIGMS to form the Drosophila Research & Screening Center-Biomedical Technology Research Resource (DRSC-BTRR). The P41-funded DRSC-BTRR (N. Perrimon, PI; S. Mohr, Co-I) builds upon and extends past goals of the Drosophila RNAi Screening Center.

As the DRSC-BTRR, we are working together with collaborators whose 'driving biomedical projects' inform development of new technologies at the DRSC. At the same time, we continue to support Drosophila cell-based RNAi and CRIPSR knockout screens and related...

Read more about DRSC-Biomedical Technology Research Resource
Photo of 384-well assay plates

Drosophila cell screen with DRSC reagent library contributes to identification of new therapeutic target for renal cancer

October 7, 2019

We here at the DRSC/TRiP are thrilled to see this study from Hilary Nicholson et al. published in Science Signaling.

The study provides a great example of how screens in Drosophila cultured cells can be used as part of a cross-species platform aimed at discovery of new targets for disease treatment. The work represents a collaboration between the laboratory of 2019 Nobel Prize winner W. Kaelin and DRSC PI N. Perrimon.

...

Read more about Drosophila cell screen with DRSC reagent library contributes to identification of new therapeutic target for renal cancer

Contact Us

Please contact us for any questions.

Publications

Justin A Bosch, Shannon Knight, Oguz Kanca, Jonathan Zirin, Donghui Yang-Zhou, Yanhui Hu, Jonathan Rodiger, Gabriel Amador, Hugo J Bellen, Norbert Perrimon, and Stephanie E Mohr. 2020. “Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes.” Curr Protoc Mol Biol, 130, 1, Pp. e112.Abstract
The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.
Raghuvir Viswanatha, Roderick Brathwaite, Yanhui Hu, Zhongchi Li, Jonathan Rodiger, Pierre Merckaert, Verena Chung, Stephanie E Mohr, and Norbert Perrimon. 2019. “Pooled CRISPR Screens in Drosophila Cells.” Curr Protoc Mol Biol, 129, 1, Pp. e111.Abstract
High-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow-up studies performed in vivo in Drosophila, mammalian cells, and/or other systems. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Pooled-format screening with Cas9-expressing Drosophila S2R+ cells in the presence of cytotoxin Support Protocol 1: Optimization of cytotoxin concentration for Drosophila cell screening Support Protocol 2: CRISPR sgRNA library design and production for Drosophila cell screening Support Protocol 3: Barcode deconvolution and analysis of screening data.
Hilary E Nicholson, Zeshan Tariq, Benjamin E Housden, Rebecca B Jennings, Laura A Stransky, Norbert Perrimon, Sabina Signoretti, and William G Kaelin. 2019. “HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species.” Sci Signal, 12, 601.Abstract
Inactivation of the tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2α (HIF-2α). HIF-2α inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. Here, we identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and inactivation in two species (human and ) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2α transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2α was not required for the increased CDK4/6 requirement of ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α-dependent ccRCC cells and not antagonistic with HIF-2α inhibition in HIF-2α-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2α inhibitors.
Stephanie E Mohr, Kirstin Rudd, Yanhui Hu, Wei R Song, Quentin Gilly, Michael Buckner, Benjamin E Housden, Colleen Kelley, Jonathan Zirin, Rong Tao, Gabriel Amador, Katarzyna Sierzputowska, Aram Comjean, and Norbert Perrimon. 12/9/2017. “Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells.” G3 (Bethesda).Abstract
Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wildtype or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and more than 1800 putative zinc-responsive genes. Analysis of the 'omics data points to the relevance of ion transporters, glutathione-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells and that rdog is transcriptionally up-regulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the 'omics datasets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.
Huajin Wang, Michel Becuwe, Benjamin E Housden, Chandramohan Chitraju, Ashley J Porras, Morven M Graham, Xinran N Liu, Abdou Rachid Thiam, David B Savage, Anil K Agarwal, Abhimanyu Garg, Maria-Jesus Olarte, Qingqing Lin, Florian Fröhlich, Hans Kristian Hannibal-Bach, Srigokul Upadhyayula, Norbert Perrimon, Tomas Kirchhausen, Christer S Ejsing, Tobias C Walther, and Robert V Farese. 2016. “Seipin is required for converting nascent to mature lipid droplets.” Elife, 5.Abstract

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

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