Cell-based RNAi (DRSC)

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Quick link to UP-TORR (view cell & in vivo reagents)

Drosophila cell-based RNAi screens have been supported by our facility since 2003. We provide reagents targeting individual genes, focused libraries, genome-scale libraries, and other resources for on-site screening at our facility or off-site screening at your institution.

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

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Publications

Eui Jae Sung, Masasuke Ryuda, Hitoshi Matsumoto, Outa Uryu, Masanori Ochiai, Molly E Cook, Na Young Yi, Huanchen Wang, James W Putney, Gary S Bird, Stephen B Shears, and Yoichi Hayakawa. 12/11/2017. “Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress.” Proc Natl Acad Sci U S A.Abstract
A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.
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.
Benjamin E Housden, Matthias Muhar, Matthew Gemberling, Charles A Gersbach, Didier YR Stainier, Geraldine Seydoux, Stephanie E Mohr, Johannes Zuber, and Norbert Perrimon. 10/31/2016. “Loss-of-function genetic tools for animal models: cross-species and cross-platform differences.” Nat Rev Genet. Publisher's VersionAbstract

Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.

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.

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.

Arunachalam Vinayagam, Meghana M Kulkarni, Richelle Sopko, Xiaoyun Sun, Yanhui Hu, Ankita Nand, Christians Villalta, Ahmadali Moghimi, Xuemei Yang, Stephanie E Mohr, Pengyu Hong, John M Asara, and Norbert Perrimon. 9/13/2016. “An Integrative Analysis of the InR/PI3K/Akt Network Identifies the Dynamic Response to Insulin Signaling.” Cell Reports, 16, 11, Pp. 3062-3074.Abstract

Insulin regulates an essential conserved signaling pathway affecting growth, proliferation, and meta- bolism. To expand our understanding of the insulin pathway, we combine biochemical, genetic, and computational approaches to build a comprehensive Drosophila InR/PI3K/Akt network. First, we map the dynamic protein-protein interaction network sur- rounding the insulin core pathway using bait-prey interactions connecting 566 proteins. Combining RNAi screening and phospho-specific antibodies, we find that 47% of interacting proteins affect pathway activity, and, using quantitative phospho- proteomics, we demonstrate that $10% of interact- ing proteins are regulated by insulin stimulation at the level of phosphorylation. Next, we integrate these orthogonal datasets to characterize the structure and dynamics of the insulin network at the level of protein complexes and validate our method by iden- tifying regulatory roles for the Protein Phosphatase 2A (PP2A) and Reptin-Pontin chromatin-remodeling complexes as negative and positive regulators of ribosome biogenesis, respectively. Altogether, our study represents a comprehensive resource for the study of the evolutionary conserved insulin network. 

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