Method or protocol

2016
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.

2015
Shuailiang Lin, Ben Ewen-Campen, Xiaochun Ni, Benjamin E Housden, and Norbert Perrimon. 2015. “In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.” Genetics, 201, 2, Pp. 433-42.Abstract

A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.

2015_Genetics_Lin.pdf Supplement.pdf Corrigendum.pdf
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.

2015_G3_Mohr.pdf Reagent Table S1.xlsx
Lizabeth A Perkins, Laura Holderbaum, Rong Tao, Yanhui Hu, Richelle Sopko, Kim McCall, Donghui Yang-Zhou, Ian Flockhart, Richard Binari, Hye-Seok Shim, Audrey Miller, Amy Housden, Marianna Foos, Sakara Randkelv, Colleen Kelley, Pema Namgyal, Christians Villalta, Lu-Ping Liu, Xia Jiang, Qiao Huan-Huan, Xia Wang, Asao Fujiyama, Atsushi Toyoda, Kathleen Ayers, Allison Blum, Benjamin Czech, Ralph Neumuller, Dong Yan, Amanda Cavallaro, Karen Hibbard, Don Hall, Lynn Cooley, Gregory J Hannon, Ruth Lehmann, Annette Parks, Stephanie E Mohr, Ryu Ueda, Shu Kondo, Jian-Quan Ni, and Norbert Perrimon. 2015. “The Transgenic RNAi Project at Harvard Medical School: Resources and Validation.” Genetics, 201, 3, Pp. 843-52.Abstract

To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

2015_Genetics_Perkins.pdf Supplement.pdf
2014
Benjamin E Housden, Shuailiang Lin, and Norbert Perrimon. 2014. “Cas9-based genome editing in Drosophila.” Methods Enzymol, 546, Pp. 415-39.Abstract

Our ability to modify the Drosophila genome has recently been revolutionized by the development of the CRISPR system. The simplicity and high efficiency of this system allows its widespread use for many different applications, greatly increasing the range of genome modification experiments that can be performed. Here, 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.

2014_Methods Enzymol_Housden.pdf
Arunachalam Vinayagam, Jonathan Zirin, Charles Roesel, Yanhui Hu, Bahar Yilmazel, Anastasia A Samsonova, Ralph A Neumüller, Stephanie E Mohr, and Norbert Perrimon. 2014. “Integrating protein-protein interaction networks with phenotypes reveals signs of interactions.” Nat Methods, 11, 1, Pp. 94-9.Abstract

A major objective of systems biology is to organize molecular interactions as networks and to characterize information flow within networks. We describe a computational framework to integrate protein-protein interaction (PPI) networks and genetic screens to predict the 'signs' of interactions (i.e., activation-inhibition relationships). We constructed a Drosophila melanogaster signed PPI network consisting of 6,125 signed PPIs connecting 3,352 proteins that can be used to identify positive and negative regulators of signaling pathways and protein complexes. We identified an unexpected role for the metabolic enzymes enolase and aldo-keto reductase as positive and negative regulators of proteolysis, respectively. Characterization of the activation-inhibition relationships between physically interacting proteins within signaling pathways will affect our understanding of many biological functions, including signal transduction and mechanisms of disease.

2014_Nat Methods_Vinayagam.pdf Supplemental Files.zip
Bahar Yilmazel, Yanhui Hu, Frederic Sigoillot, Jennifer A Smith, Caroline E Shamu, Norbert Perrimon, and Stephanie E Mohr. 2014. “Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis.” BMC Bioinformatics, 15, Pp. 192.Abstract

BACKGROUND: RNA interference (RNAi) is an effective and important tool used to study gene function. For large-scale screens, RNAi is used to systematically down-regulate genes of interest and analyze their roles in a biological process. However, RNAi is associated with off-target effects (OTEs), including microRNA (miRNA)-like OTEs. The contribution of reagent-specific OTEs to RNAi screen data sets can be significant. In addition, the post-screen validation process is time and labor intensive. Thus, the availability of robust approaches to identify candidate off-targeted transcripts would be beneficial. RESULTS: Significant efforts have been made to eliminate false positive results attributable to sequence-specific OTEs associated with RNAi. These approaches have included improved algorithms for RNAi reagent design, incorporation of chemical modifications into siRNAs, and the use of various bioinformatics strategies to identify possible OTEs in screen results. Genome-wide Enrichment of Seed Sequence matches (GESS) was developed to identify potential off-targeted transcripts in large-scale screen data by seed-region analysis. Here, we introduce a user-friendly web application that provides researchers a relatively quick and easy way to perform GESS analysis on data from human or mouse cell-based screens using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), as well as for Drosophila screens using shRNAs. Online GESS relies on up-to-date transcript sequence annotations for human and mouse genes extracted from NCBI Reference Sequence (RefSeq) and Drosophila genes from FlyBase. The tool also accommodates analysis with user-provided reference sequence files. CONCLUSION: Online GESS provides a straightforward user interface for genome-wide seed region analysis for human, mouse and Drosophila RNAi screen data. With the tool, users can either use a built-in database or provide a database of transcripts for analysis. This makes it possible to analyze RNAi data from any organism for which the user can provide transcript sequences.

2014_BMC Bioinfo_Yilmazel.pdf
2013
Clément Carré, Caroline Jacquier, Anne-Laure Bougé, Fabrice de Chaumont, Corinne Besnard-Guerin, Hélène Thomassin, Josette Pidoux, Bruno Da Silva, Eleftheria Chalatsi, Sarah Zahra, Jean-Christophe Olivo-Marin, Hélène Munier-Lehmann, and Christophe Antoniewski. 2013. “AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.” PLoS One, 8, 9, Pp. e74296.Abstract

Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.

2013_PLOS One_Carre.pdf Supplemental Files.zip
Max V Staller, Dong Yan, Sakara Randklev, Meghan D Bragdon, Zeba B Wunderlich, Rong Tao, Lizabeth A Perkins, Angela H Depace, and Norbert Perrimon. 2013. “Depleting gene activities in early Drosophila embryos with the "maternal-Gal4-shRNA" system.” Genetics, 193, 1, Pp. 51-61.Abstract

In a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal-zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes.

2013_Genetics_Staller.pdf Table S1.pdf
Yanhui Hu, Richelle Sopko, Marianna Foos, Colleen Kelley, Ian Flockhart, Noemie Ammeux, Xiaowei Wang, Lizabeth Perkins, Norbert Perrimon, and Stephanie E Mohr. 2013. “FlyPrimerBank: an online database for Drosophila melanogaster gene expression analysis and knockdown evaluation of RNAi reagents.” G3 (Bethesda), 3, 9, Pp. 1607-16.Abstract

The evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo.

2013_G3_Hu.pdf Supplemental Files.zip
Xingjie Ren, Jin Sun, Benjamin E Housden, Yanhui Hu, Charles Roesel, Shuailiang Lin, Lu-Ping Liu, Zhihao Yang, Decai Mao, Lingzhu Sun, Qujie Wu, Jun-Yuan Ji, Jianzhong Xi, Stephanie E Mohr, Jiang Xu, Norbert Perrimon, and Jian-Quan Ni. 2013. “Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.” Proc Natl Acad Sci U S A, 110, 47, Pp. 19012-7.Abstract

The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

2013_PNAS_Ren.pdf Supplement.pdf
Arunachalam Vinayagam, Yanhui Hu, Meghana Kulkarni, Charles Roesel, Richelle Sopko, Stephanie E Mohr, and Norbert Perrimon. 2013. “Protein complex-based analysis framework for high-throughput data sets.” Sci Signal, 6, 264, Pp. rs5.Abstract

Analysis of high-throughput data increasingly relies on pathway annotation and functional information derived from Gene Ontology. This approach has limitations, in particular for the analysis of network dynamics over time or under different experimental conditions, in which modules within a network rather than complete pathways might respond and change. We report an analysis framework based on protein complexes, which are at the core of network reorganization. We generated a protein complex resource for human, Drosophila, and yeast from the literature and databases of protein-protein interaction networks, with each species having thousands of complexes. We developed COMPLEAT (http://www.flyrnai.org/compleat), a tool for data mining and visualization for complex-based analysis of high-throughput data sets, as well as analysis and integration of heterogeneous proteomics and gene expression data sets. With COMPLEAT, we identified dynamically regulated protein complexes among genome-wide RNA interference data sets that used the abundance of phosphorylated extracellular signal-regulated kinase in cells stimulated with either insulin or epidermal growth factor as the output. The analysis predicted that the Brahma complex participated in the insulin response.

2013_Sci Sig_Vinayagam.pdf Supplemental Files.zip
Yanhui Hu, Charles Roesel, Ian Flockhart, Lizabeth Perkins, Norbert Perrimon, and Stephanie E Mohr. 2013. “UP-TORR: online tool for accurate and Up-to-Date annotation of RNAi Reagents.” Genetics, 195, 1, Pp. 37-45.Abstract

RNA interference (RNAi) is a widely adopted tool for loss-of-function studies but RNAi results only have biological relevance if the reagents are appropriately mapped to genes. Several groups have designed and generated RNAi reagent libraries for studies in cells or in vivo for Drosophila and other species. At first glance, matching RNAi reagents to genes appears to be a simple problem, as each reagent is typically designed to target a single gene. In practice, however, the reagent-gene relationship is complex. Although the sequences of oligonucleotides used to generate most types of RNAi reagents are static, the reference genome and gene annotations are regularly updated. Thus, at the time a researcher chooses an RNAi reagent or analyzes RNAi data, the most current interpretation of the RNAi reagent-gene relationship, as well as related information regarding specificity (e.g., predicted off-target effects), can be different from the original interpretation. Here, we describe a set of strategies and an accompanying online tool, UP-TORR (for Updated Targets of RNAi Reagents; www.flyrnai.org/up-torr), useful for accurate and up-to-date annotation of cell-based and in vivo RNAi reagents. Importantly, UP-TORR automatically synchronizes with gene annotations daily, retrieving the most current information available, and for Drosophila, also synchronizes with the major reagent collections. Thus, UP-TORR allows users to choose the most appropriate RNAi reagents at the onset of a study, as well as to perform the most appropriate analyses of results of RNAi-based studies.

2013_Genetics_Hu.pdf Supplement.pdf
2012
Marcelo Perez-Pepe, Victoria Slomiansky, Mariela Loschi, Luciana Luchelli, Maximiliano Neme, María Gabriela Thomas, and Graciela Lidia Boccaccio. 2012. “BUHO: a MATLAB script for the study of stress granules and processing bodies by high-throughput image analysis.” PLoS One, 7, 12, Pp. e51495.Abstract

The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny.

2012_PLOS One_Perez-Pepe.pdf Supplemental Files.zip
2011
Matthew Booker, Anastasia A Samsonova, Young Kwon, Ian Flockhart, Stephanie E Mohr, and Norbert Perrimon. 2011. “False negative rates in Drosophila cell-based RNAi screens: a case study.” BMC Genomics, 12, Pp. 50.Abstract

BACKGROUND: High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention. RESULTS: We performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene. CONCLUSIONS: RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.

2011_BMCGenomics_Booker.pdf Supplement 1.xls Supplement 2.xls
Yanhui Hu, Ian Flockhart, Arunachalam Vinayagam, Clemens Bergwitz, Bonnie Berger, Norbert Perrimon, and Stephanie E Mohr. 2011. “An integrative approach to ortholog prediction for disease-focused and other functional studies.” BMC Bioinformatics, 12, Pp. 357.Abstract

BACKGROUND: Mapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward. RESULTS: We report a simple but effective tool, the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT; http://www.flyrnai.org/diopt), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist). CONCLUSIONS: DIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.

2011_BMC Bioinfo_Hu.pdf Supplemental Files.zip
Norbert Perrimon, Jonathan Zirin, and Jianwu Bai. 2011. “Primary cell cultures from Drosophila gastrula embryos.” J Vis Exp, 48.Abstract

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

2011_J Vis Exp_Perrimon.pdf
2010
Chaohong Wu, Joost Schulte, Katharine J Sepp, Troy J Littleton, and Pengyu Hong. 2010. “Automatic robust neurite detection and morphological analysis of neuronal cell cultures in high-content screening.” Neuroinformatics, 8, 2, Pp. 83-100.Abstract

Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutamine-mediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington's Disease (HD) model.

2010_Neuroinfor_Wu.pdf
Michael Schnall-Levin, Yong Zhao, Norbert Perrimon, and Bonnie Berger. 2010. “Conserved microRNA targeting in Drosophila is as widespread in coding regions as in 3'UTRs.” Proc Natl Acad Sci U S A, 107, 36, Pp. 15751-6.Abstract

MicroRNAs (miRNAs) are a class of short noncoding RNAs that regulate protein-coding genes posttranscriptionally. In animals, most known miRNA targeting occurs within the 3'UTR of mRNAs, but the extent of biologically relevant targeting in the ORF or 5'UTR of mRNAs remains unknown. Here, we develop an algorithm (MinoTar-miRNA ORF Targets) to identify conserved regulatory motifs within protein-coding regions and use it to estimate the number of preferentially conserved miRNA-target sites in ORFs. We show that, in Drosophila, preferentially conserved miRNA targeting in ORFs is as widespread as it is in 3'UTRs and that, while far less abundant, conserved targets in Drosophila 5'UTRs number in the hundreds. Using our algorithm, we predicted a set of high-confidence ORF targets and selected seven miRNA-target pairs from among these for experimental validation. We observed down-regulation by the miRNA in five out of seven cases, indicating our approach can recover functional sites with high confidence. Additionally, we observed additive targeting by multiple sites within a single ORF. Altogether, our results demonstrate that the scale of biologically important miRNA targeting in ORFs is extensive and that computational tools such as ours can aid in the identification of such targets. Further evidence suggests that our results extend to mammals, but that the extent of ORF and 5'UTR targeting relative to 3'UTR targeting may be greater in Drosophila.

2010_PNAS_Schnall-Levin.pdf Supplement.pdf
2009
Jianwu Bai, Katharine J Sepp, and Norbert Perrimon. 2009. “Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.” Nat Protoc, 4, 10, Pp. 1502-12.Abstract

We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes approximately 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.

2009_Nat Prot_Bai.pdf

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