Cell-based RNAi

Chen X and Xu L. 2016. “Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway.” Methods in Molecular Biology. Publisher's VersionAbstract

The transforming growth factor-β (TGF-β) family of cytokines figures prominently in regulation of embryonic development and adult tissue homeostasis from Drosophila to mammals. Genetic defects affecting TGF-β signaling underlie developmental disorders and diseases such as cancer in human. Therefore, delineating the molecular mechanism by which TGF-β regulates cell biology is critical for understanding normal biology and disease mechanisms. Forward genetic screens in model organisms and biochemical approaches in mammalian tissue culture were instrumental in initial characterization of the TGF-β signal transduction pathway. With complete sequence information of the genomes and the advent of RNA interference (RNAi) technology, genome-wide RNAi screening emerged as a powerful functional genomics approach to systematically delineate molecular components of signal transduction pathways. Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined >90 % of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed.

Yanhui Hu, Aram Comjean, Charles Roesel, Arunachalam Vinayagam, Ian Flockhart, Jonathan Zirin, Lizabeth Perkins, Norbert Perrimon, and Stephanie E Mohr. 10/11/2016. “FlyRNAi.org—the database of the Drosophila RNAi screening center and transgenic RNAi project: 2017 update.” Nucleic Acids Research. Publisher's VersionAbstract

The FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches.

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.

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. 

Joel M Swenson, Serafin U Colmenares, Amy R Strom, Sylvain V Costes, and Gary H Karpen. 2016. “The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic.” Elife, 5.Abstract

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.

Screenshot of the Online Tools Overview page

Is it a hit? On mining our data sets.

July 22, 2016

The DRSC/TRiP-FGR's FlyRNAi database stores results from the many cell-based screens done since 2003 using DRSC Drosophila RNAi libraries. It also stores information about knockdown and phenotypes resulting from specific combinations of in vivo RNAi fly stocks (including our TRiP stocks and also VDRC and NIG-Japan stocks). The in vivo data includes directly deposited data and results curated by FlyBase from the literature.

Even if you are not interested to do a fly RNAi screen, these data might help you. For...

Read more about Is it a hit? On mining our data sets.
2016 Sep 23

Boston Area Drosophila Meeting

1:00pm to 4:30pm

Location: 

University of Massachusetts Boston

The DRSC-Functional Genomics Resources (formerly DRSC & TRiP) will be participating in the Boston Area Drosophila Meeting, which was organized by Alexey Verakas of UMass Boston and Jim Walker of Harvard Medical School. Hear about what's new in technologies and online tools at this regional meeting of experts in Drosophila research.

Search results for the term oogenesis at the Drosophila protocols portal

Beta-testing a "Drosophila Protocols Portal"

June 16, 2016

The DRSC-FGR has developed a beta version of a database and online search for protocols, the Drosophila Protocols Portal, relevant to Drosophila research. The goal is to provide a central portal for protocols distributed across the web. We collected protocols from protocol databases, lab websites, YouTube, Drosophila Information Service (DIS), and relevant journals. You can view the results by topic or search for specific terms.

Longer-term goals...

Read more about Beta-testing a "Drosophila Protocols Portal"
2016 Sep 28

Functional genomics techniques in Drosophila and their potential application in non-model insects

11:45am to 12:00pm

Location: 

Orlando, FL

DRSC-FGR Director S. Mohr will be presenting in the symposium Insect Genetic Technologies: State of the Art and Promise for the Future at the International Congress of Entomology (ICE 2016). Come hear what is possible in Drosophila that might be applied to other insect species. Wednesday, September 28, 2016 at 11:45 am (symposium from 9:30 - 12:30).

Ian T Flockhart, Matthew Booker, Yanhui Hu, Benjamin McElvany, Quentin Gilly, Bernard Mathey-Prevot, Norbert Perrimon, and Stephanie E Mohr. 2012. “FlyRNAi.org--the database of the Drosophila RNAi screening center: 2012 update.” Nucleic Acids Res, 40, Database issue, Pp. D715-9.Abstract

FlyRNAi (http://www.flyrnai.org), the database and website of the Drosophila RNAi Screening Center (DRSC) at Harvard Medical School, serves a dual role, tracking both production of reagents for RNA interference (RNAi) screening in Drosophila cells and RNAi screen results. The database and website is used as a platform for community availability of protocols, tools, and other resources useful to researchers planning, conducting, analyzing or interpreting the results of Drosophila RNAi screens. Based on our own experience and user feedback, we have made several changes. Specifically, we have restructured the database to accommodate new types of reagents; added information about new RNAi libraries and other reagents; updated the user interface and website; and added new tools of use to the Drosophila community and others. Overall, the result is a more useful, flexible and comprehensive website and database.

Clemens Bergwitz, Mark J Wee, Sumi Sinha, Joanne Huang, Charles DeRobertis, Lawrence B Mensah, Jonathan Cohen, Adam Friedman, Meghana Kulkarni, Yanhui Hu, Arunachalam Vinayagam, Michael Schnall-Levin, Bonnie Berger, Lizabeth A Perkins, Stephanie E Mohr, and Norbert Perrimon. 2013. “Genetic determinants of phosphate response in Drosophila.” PLoS One, 8, 3, Pp. e56753.Abstract

Phosphate is required for many important cellular processes and having too little phosphate or too much can cause disease and reduce life span in humans. However, the mechanisms underlying homeostatic control of extracellular phosphate levels and cellular effects of phosphate are poorly understood. Here, we establish Drosophila melanogaster as a model system for the study of phosphate effects. We found that Drosophila larval development depends on the availability of phosphate in the medium. Conversely, life span is reduced when adult flies are cultured on high phosphate medium or when hemolymph phosphate is increased in flies with impaired malpighian tubules. In addition, RNAi-mediated inhibition of MAPK-signaling by knockdown of Ras85D, phl/D-Raf or Dsor1/MEK affects larval development, adult life span and hemolymph phosphate, suggesting that some in vivo effects involve activation of this signaling pathway by phosphate. To identify novel genetic determinants of phosphate responses, we used Drosophila hemocyte-like cultured cells (S2R+) to perform a genome-wide RNAi screen using MAPK activation as the readout. We identified a number of candidate genes potentially important for the cellular response to phosphate. Evaluation of 51 genes in live flies revealed some that affect larval development, adult life span and hemolymph phosphate levels.

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