Core Facilities - Annual Report 2014 by EPFL School of Life Sciences

EPFL School of Life Sciences Core Facilities and Technology Platforms Report 2014

EPFL School of Life Sciences - 2013 Annual Report

Core Facilities & Technology Platforms

In addition, scientists from our School of Life Sciences closely collaborate with other services in the Lemanic region, including the ‘Center for Biomedical Imaging’ (http://www.cibm.ch) and the ‘Lausanne Genomics Technologies Facility’ (http://www.unil.ch/gtf/en/home.html) The following pages describe the Life Sciences-related core facilities and technology platforms currently available at the EPFL School of Life Sciences.

159

Core Facilities & Technology Platforms

In its goal to offer maximal support to its students and scientists in their training and research capabilities, EPFL and its School of Life Sciences have made a significant investment over the past years to establish stateof-the-art technology platforms and core facilities. These facilities are directed and managed by dedicated teams of highly trained and experienced staff and are run on a fee-for-service basis. They offer training, access to technology, assistance with experimental design and high level data analysis, and collaborations. The platforms are also involved in the School’s undergraduate and graduate teaching programs.

EPFL School of Life Sciences - 2014 Annual Report

Bioelectron Microscopy - BioEM http://BioEM.epfl.ch

Introduction

The BioEM Facility provides life scientists at the EPFL and the lemanic area with the equipment and expertise for imaging biological samples with electron microscopy. This platform has a wide range of techniques available with both transmission and scanning electron microscopy using preparation techniques at either cryo or ambient temperatures.

Services and Technologies

The facility offers training and services in the following methods: • Transmission electron microscopy • Cryo transmission electron microscopy • Scanning electron microscopy • Focussed ion beam scanning electron microscopy • Correlated light and electron microscopy • Pre-embedding immunolabelling • Post-embedding immuno labelling • Single particle imaging and analysis • Cryo fixation with high pressure freezing • Standard chemical fixation methods including microwave

Image: Transmission electron microscograph showing mitochondria and the endoplasmic reticulum in an heptocyte of a young rat.

Selected Publications » Guichard, P., Desfosses, A., Maheshwari, A., Hachet, V., Dietrich, C., Brune, A., Ishikawa, T., Sachse, C., and Gönczy, P. (2012). Cartwheel architecture of Trichonympha basal body. Science. 337(6094):553. » Pino, E., Amamoto, R., Zheng, L., Cacquevel, M., Sarria, J.C., Knott, G.W., Schneider, B.L. (2013) FOXO3 determines the accumulation of α-synuclein and controls the fate of dopaminergic neurons in the substantia nigra. Hum Mol Genet. 23(6):1435-52. » von Tobel L, Mikeladze-Dvali T, Delattre M, Balestra FR, Blanchoud S, Finger S, Knott G, Müller-Reichert T, Gönczy P. (2014). SAS-1 is a C2 domain protein critical for centriole integrity in C. elegans. PLoS Genet. 10(11). » Maco, B., Cantoni, M., Holtmaat, A., Kreshuk, A., Hamprecht, F.A., Knott, G.W. (2014). Semiautomated correlative 3D electron microscopy of E. coli-imaged axons and dendrites. Nat Protoc. 2014;9(6):1354-66

160

Team Members Facility Head Graham Knott

Senior Scientist Davide Demurtas Scientific Assistant Catherine Maclachlan Technicians Stéphanie Clerc-Rosset, Mary-Claude Croisier-Coeytaux Adminstrative Assistant Annick Evequoz Contact Information Graham Knott EPFL SV PTBIOEM AI 0143 Station 19 CH-1015 Lausanne Tel: +41 (0) 21 693 18 62 graham.knott@epfl.ch

EPFL School of Life Sciences - 2014 Annual Report

BioImaging & Optics - BIOP http://biop.epfl.ch/

The Bioimaging and Optics platform (BIOP) located in the faculty of Life Science (SV) provides access to state of the art light microscopes and important expertise to solve challenging (biological) questions with modern light-microscopy. A broad range of instruments ranging from simple wide-field imaging systems over standard point-scanning confocal microscopes up to a high-end 2-Photon-excitation microscope and microscopes which allow imaging below the diffraction limit (high/ super resolution microscopes) are currently available. Scientists who want to make use of the available equipment are trained so that they can use the instruments either independently or under the supervision of the BIOP team. The competence in sample preparation and image acquisition is complemented with strong image processing skills and necessary computer power to perform image processing. The idea is to link the image analysis with the image acquisition as early as possible in order to guarantee optimal scientific results.

Services and Technologies • Wide-field transmission and fluorescent microscopes • Life cell imaging microscopes • Single and multiple-beam confocal microscopes • 2P microscope • Fluorescence Lifetime Microscopy (FLIM) • High resolution and super resolution microscopes • (SIM, STROM, STED) • Image Processing tools (commercially available and/or custom built)

Team Members Facility Head Arne Seitz

Microscopy Jose Artacho Luigi Bozzo Thierry Laroche Image Processing Olivier Burri Romain Guiet Administrative Assistant Maureen Hersperger Contact Information Arne Seitz EPFL SV PTBIOP AI-0241 Station 15, CH-1015 Lausanne Tel: +41 (0) 21 693 96 18 Fax: +41 (0) 21 693 95 85 arne.seitz@epfl.ch

Selected Publications » Esser-von Bieren J, Mosconi I, Guiet R, Piersgilli A, Volpe B, Chen F, Gause WC, Seitz A, Verbeek JS, Harris NL. (2013) Antibodies trap tissue migrating helminth larvae and prevent tissue damage by driving IL-4Rαindependent alternative differentiation of macrophages. PLoS Pathog. 9(11): e1003771. » Lukinavičius G, Reymond L, D’Este E, Masharina A, Göttfert F, Ta H, Güther A, Fournier M, Rizzo S, Waldmann H, Blaukopf C, Sommer C, Gerlich DW, Arndt HD, Hell SW, Johnsson K. (2014) Fluorogenic probes for livecell imaging of the cytoskeleton. Nat Methods. 11(7):731-3. » Virginie Hamel, Paul Guichard, Mathias Fournier, Romain Guiet, Isabelle Flückiger, Arne Seitz, and Pierre Gönczy (2014) Correlative multicolor 3D SIM and STORM microscopy. Biomedical Optics Express 5(10): 3326-3336. » Burri O., Guiet R., Seitz A. (2014) Development of freely available software tools from the perspective of a multi user core facility. Microscopy: advances in scientific research and education 2 (13): 978-84.

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Core Facilities & Technology Platforms

Introduction

EPFL School of Life Sciences - 2014 Annual Report

Bioinformatics & Biostatisitcs - BBCF http://bbcf.epfl.ch

Introduction

The Bioinformatics and Biostatistics Core Facility (BBCF) provides the EPFL and Lemanic institutions with extensive support in bioinformatics and biostatistics, from designing experiments to interpreting and visualizing complex data. Its main competences are in management and analysis of genomic data, mathematical modeling and statistical analysis of quantitative biological data. The facility works in close relationship with the Geneva and Lausanne Genomics platforms and complements their respective bioinformatics team with additional support for the analysis of large or complex data sets, for the implementation of data processing pipelines for new highthroughput technologies, and for the statistical planning in complex experimental designs. It also helps researchers with mining public databases, designing and setting up local databases, inferring mathematical models from experimental data and running simulations to validate a model. The facility acts as a point of contact between the experimental biologists and the research groups in bioinformatics and in basic sciences. It also makes the junction between the EPFL Life Science community and the various resources maintained by the Swiss Institute of Bioinformatics, in particular the Vital-IT high performance computing center.

Services and Technologies • Analysis of high-throughput sequencing data: http://htsstation.epfl.ch • Management of genomics data http://biorepo.epfl.ch • Software development for genomics and bioinformatics: https://github.com/bbcf and http://bbcftools.epfl.ch • Building blocks for online genomic data manipulations: http://gdv.epfl.ch/bs • Databases and tools for lipidomics: http://lipidomes.org • Atlas of the Drosophila midgut: http://flygut.epfl.ch • Database of PCR primers http://bbcftools.epfl.ch/primerviz • Database of palmitoylated proteins: http://swisspalm.epfl.ch

Selected Publications » » » » »

David, F. P. A. et al. (2014). HTSstation: A Web Application and Open-Access Libraries for High-Throughput Sequencing Data Analysis. PLoS ONE 9:e85879. Porro, A. et al. (2014). Functional characterization of the TERRA transcriptome at damaged telomeres. Nat Commun 5:5379. Steijger, T. et al. (2013). Assessment of transcript reconstruction methods for RNA-seq. Nat Methods 10:1177–1184. Andrey, G. et al. (2013). A switch between topological domains underlies HoxD genes collinearity in mouse limbs. Science 340:1234167. Buchon, N. et al. (2013). Morphological and molecular characterization of adult midgut compartmentalization in Drosophila. Cell Rep 3:1725–1738.

162

Team Members Facility Head Jacques Rougemont

Postdoctoral Researchers Fabrice David Philippe Jacquet Marion Leleu Scientific Assistants Sara Benmohammed Julien Delafontaine Yohan Jarosz Yoann Mouscaz Adminstrative Assistant Lisa Cessy Contact Information Dr. Jacques Rougemont EPFL SV PTBB AAB 025, Station 15 CH-1015 Lausanne +41 (0) 21 693 9573 jacques.rougemont@epfl.ch

EPFL School of Life Sciences - 2014 Annual Report

Biomolecular Screening - BSF http://bsf.epfl.ch/

The Biomolecular Screening Facility is a multidisciplinary platform created in 2006 at the EPFL for performing screens in life sciences-related projects. In the frame of the NCCR-Chemical Biology, the BSF is hosting ACCESS, Academic Chemical Screening Platform for Switzerland, that provides chemical diversity, screening facilities and know-how in chemical genetics for academics countrywide.

Services and Technologies • Access to instrumentation dedicated to microplates and cell culture facilities • Chemical libraries of about 100’000 small molecules (BSF-ACCESS)

• Pilot screenings & Primary screening campaigns • Hits confirmation, Dose response assays & Secondary screens

• Genome–wide siRNA collections

• High Content Screening. Phenotypic assays by Digital Holographic Imaging (label free) and Fluorescence Microscopy

• Compound storage and management

• Image processing for high content screening read-outs

• Compounds delivery to partners • Assay development and validation for HTS

• Data management using in house developed Laboratory Implementation Management System (LIMS).

• Assay automation and statistical validations

• Cheminformatics

Team Members Facility Head Gerardo Turcatti Scientists Damiano Banfi Marc Chambon Fabien Kuttler Postdoctoral Researchers Benjamin Rappaz Marianne Seijo Assistants Nathalie Ballanfat Julien Bortoli Gérald Cruciani Antoine Gibelin Administrative Assistant Maureen Hersperger Contact Information Gerardo Turcatti, PhD, MER EPFL SV PTCB AAB 003, Station 15 CH-1015 Lausanne Tel: +41(0) 21 693 9666 gerardo.turcatti@epfl.ch

Selected Publications » Turcatti, G. (2014) Developing the Biomolecular Screening Facility at the EPFL into the Chemical Biology Screening Platform for Switzerland, Comb Chem High T Scr 17: 369-376. » Röhrig, U. F., Majjigapu, S. R., Chambon, M., Bron, S., Pilotte, L., Colau, D., Van den Eynde, B. J., Turcatti, G., Vogel, P., Zoete, V., and Michielin, O. (2014) Detailed analysis and follow-up studies of a high-throughput screening for indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, European Journal of Medicinal Chemistry 84: 284-301. » Rappaz, B., Breton, B., Shaffer, E., and Turcatti, G. (2014) Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening, Comb Chem High T Scr 17: 80-88. » Makhlouf Brahmi, M., Portmann, C., D’Ambrosio, D., Woods, T. M., Banfi, D., Reichenbach, P., Da Silva, L., Baudat, E., Turcatti, G., Lingner, J., and Gademann, K. (2013) Telomerase Inhibitors from Cyanobacteria: Isolation and Synthesis of Sulfoquinovosyl Diacylglycerols from Microcystis aeruguinosa PCC 7806, Chemistry – A European Journal 19: 4596-4601. » Kuhn, J., Shaffer, E., Mena, J., Breton, B., Parent, J., Rappaz, B., Chambon, M., Emery, Y., Magistretti, P., Depeursinge, C., Marquet, P., and Turcatti, G. (2013) Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy, ASSAY and Drug Development Technologies 11: 101-107.

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Core Facilities & Technology Platforms

Introduction

EPFL School of Life Sciences - 2014 Annual Report

Flow Cytometry - FCCF http://fccf.epfl.ch/

Introduction

Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity. Sorting allows us to capture and collect theses particles of interest for further analysis. The Flow Cytometry Core Facility (FCCF) is located in the School of Life Sciences (SV) at the Ecole Polytechnique Fédérale de Lausanne (EPFL) and is part of a network of the core facilities at the school. FCCF’s mission is to provide to all investigators at EPFL and outside, comprehensive flow cytometric analysis and sorting including instrumentation, technical and professional assistance, training and consultation. The FCCF facility aims to keep on the cutting edge of cytometric technology by constantly updating hardware. New techniques are introduced according to the user’s needs. All the facility staff are members of the ISAC society. Currently the Flow Cytometry Core Facility is equipped with seven state of the art self-service bench top analyzers as well as two high-speed BLS2 cell sorters. The Core Facility also operates an automated immunomagnetic bead cell separator from Miltenyi Biotec MACS® Technology. Researchers have the option, once trained, of performing their own acquisition/analysis or utilizing the expertise of the facility’s staff to run their samples with them.

Equipment • 1 - LSRII (Becton Dickinson) 5 lasers bench top analyser with 20 parameters. • 1 - LSRII (Becton Dickinson) 4 lasers bench top analyser with 15 parameters. • 3 - Cyan ADP (Beckman Coulter) 3 laser bench top analyser with 11 parameters. • 1 - Galios (Beckman Coulter) is a 3 laser bench top analyser with 12 parameters. • 1 - Accuri C6 is a 2 laser bench top analyser with 6 parameters and a plate reader • 1 - AutoMACS Pro is a fully automated bench-top magnetic bead sorter that can be used to perform sterile bulk sorts. • 1 - FACSAria (Becton Dickinson) 4-ways high-speed BLS-2 cell sorter, 5 lasers with 20 parameters and ACDU. • 1 - MoFlo Astrios (Beckman Coulter) 6-ways high-speed BLS-2 cell sorter, 4-laser with 19 parameters.

Services • Cell sorting • User training (machines and software) • Flow Cytometry teaching • Advice on experimental design • Setup and optimization of flow cytometry protocols • Expertise and advice include consultation on flow cytometry acquisition and data analysis • Support, in reporting flow cytometry data for manuscript and grant applications

164

Team Members Facility Head Miguel Garcia

Cytometry Operators Valérie Glutz Loïc Tauzin Adminstrative Assistant Maureen Hersperger Contact Information Miguel Garcia EPFL SV PTCF AI 0147, Station 15 CH – 1015 Lausanne Tel: +41 (0) 21 693 0901 miguel.garcia@epfl.ch https://twitter.com/Cytometry_ EPFL

EPFL School of Life Sciences - 2014 Annual Report

Histology - HCF & Comparative Pathology http://hcf.epfl.ch

Histology involves the use of a set of techniques to examine the morphology, architecture and composition of tissues. The tissue samples are processed for the study of structures seen under the microscope, also called microscopic anatomy, as opposed to gross anatomy which involves structures that can be observed with the naked eye. The histology core facility is a competence pole which provides expertise in those analyses as well as routine work for researchers. All the techniques would be worthless without the expertise of a specialist in veterinarian pathology helping researchers to analyze their slides.

Services and Technologies

The facility assists researchers in the setting up and optimizing of histological approaches specific for each scientific project. Members of the SV faculty can be trained on the available instruments like microtomes or cryostats and have then access to them for their own experiments. Also facility technicians are available to perform work for researchers: • Tissue processing to frozen, paraffin or resin sections • Histological stains like the standard Hematoxyline and eosin and special stains like Sirius red among others both manually or automated (Prisma from Sakura) • Setup and optimization of immunohistochemistry and immunofluorescence protocols manually as well as automated (Ventana Discovery xT)

• Tests of newly available techniques (RNAscope from ACDbio) or useful published methods (double immunofluorescence for two primary antibodies of the same species using TSA technology). Pathology service Pathology support is provided by a specialist who has formal postgraduate training in veterinary anatomic pathology officially acknowledged by board certification of specialty. The pathologist is trained to diagnose, describe and interpret morphologic macro- and microscopic lesions within organs and tissues. Appropriate interpretation implies proper recognition of tissue abnormalities and sound knowledge of pathologic processes of diseases that manifest as morphologic changes as well as of the experimental design and settings. The service provides the following activities: • Consulting - at the study design level for issues related to pathology investigation • Morphologic phenotyping - whole body or organ targeted for genetically engineered animals • Analysis of histological specimens • Support - in reporting pathology data for manuscript submission and grant application • Diagnostics - Post mortem examination of diseased animals within the colony.

Team Members

Facility Head Jessica Sordet-Dessimoz Collaborators Gian-Filippo Mancini Nathalie Müller Agnès Hautier Vanessa Mack Comparative Pathologist Alessandra Piersigilli Administrative Assistant Maureen Hersperger Contact Information Jessica Sordet-Dessimoz EPFL SV PTH AI0342, Station19 CH-1015 Lausanne +41 (0) 21 6930962 info.hcf@epfl.ch

• Detection of mRNA and miRNA using cold probes on the Discovery xT from Roche-Ventana Selected Publications » Lo Sasso G, Menzies KJ, Mottis A, Piersigilli A, Perino A, Yamamoto H, Schoonjans K, Auwerx J. (2014). SIRT2 deficiency modulates macrophage polarization and susceptibility to experimental colitis. PLoS One. 9(7):e103573. » Lo Sasso G, Ryu D, Mouchiroud L, Fernando SC, Anderson CL, Katsyuba E, Piersigilli A, Hottiger MO, Schoonjans K, Auwerx J. (2014). Loss of Sirt1 function improves intestinal anti-bacterial defense and protects from colitisinduced colorectal cancer. PLoS One. 9(7):e102495. » Esser-von Bieren J, Mosconi I, Guiet R, Piersigilli A, Volpe B, Chen F, Gause WC, Seitz A, Verbeek JS, Harris NL. (2013). Antibodies trap tissue migrating helminth larvae and prevent tissue damage by driving IL-4Rα-independent alternative differentiation of macrophages. PLoS Pathog. 9(11):e1003771.

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Core Facilities & Technology Platforms

Introduction

EPFL School of Life Sciences - 2014 Annual Report

Proteomics - PCF http://pcf-ptp.epfl.ch/

Introduction

In the last 10 years mass spectrometry based protein analysis has become an invaluable tool in the arsenal of techniques offered to the biologist to study the proteome, the expressed and active part of the genome. The rapid evolution of the technique has been tightly bound to the continuous increase in performance of mass spectrometers. Nowadays it is possible to get quantitative information about thousands of proteins in one experiment. Researchers can begin to think more globally. But there is still room for very detailed studies on single proteins especially those modified by post-translational modifications. The EPFL Proteomics Core Facility is a technological platform that has been created to address these needs and help researchers in using these techniques.

Services and Technologies

Instrumentation The PCF-PTP laboratory is currently equipped with sample preparation and fractionation devices (HPLC, FPLC, pI) and several mass spectrometers coupled to liquid chromatography: 3 Orbitraps, 2 QQQ LC-ESIMS/MS and 1 MALDI-TOF/TOF instruments. A mass cytometry instrument (CyTOF 2) jointly operated with the Flow Cytometry platform (PTCF) will soon complete the instrument base. The bioinformatics analysis pipeline includes Mascot, Xtandem! SEQUEST and Peaks servers for matching MS data with protein sequence databases and data post-treatment tools like Maxquant, Perseus, Pro-

teome Discoverer, PinPoint and Scaffold for protein identification, validation and pipelining of quantitative studies. Services The PCF-PTP has implemented several complementary workflows for protein analysis and offers an increasing palette of services... • Protein/Peptide Molecular Weight Measurements by Mass Spectrometry. • Mass Spectrometry based Protein/Peptide Identification from Gel or Solution. • Protein Relative Quantification by SILAC or Label-free Quantitative Analysis on collaborative basis. • Protein separation by FPLC and HPLC.

• Accurate protein quantification by SRM-MRM. • Localization and quantification of PTM’s other than phosphorylation. mixtures

profiling

and

targeted

quantification

... entertains tight collaboration with other proteomics facilities (UNILPAF, UNIGE-PCF, UNIBE) and with computer science and bioinformatics research centers (Vital-IT, SIB, etc..).

Selected Publications » Chopra, T., Hamelin, R., Armand, F., Chiappe, D., Moniatte, M., and McKinney, J.D. (2014). Quantitative Mass Spectrometry Reveals Plasticity of Metabolic Networks in Mycobacterium smegmatis. Mol. Cell. Proteomics 13, 3014–3028. » Grolimund, L., Aeby, E., Hamelin, R., Armand, F., Chiappe, D., Moniatte, M., and Lingner, J. (2013). A quantitative telomeric chromatin isolation protocol identifies different telomeric states. Nat. Commun. 4, 2848 » Schmid, A.W., Fauvet, B., Moniatte, M., and Lashuel, H.A. (2013). Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies. Mol. Cell. Proteomics 12, 3543–3558. » Simicevic, J., Schmid, A.W., Gilardoni, P.A., Zoller, B., Raghav, S.K., Krier, I., Gubelmann, C., Lisacek, F., Naef, F., Moniatte, M., and Deplancke B. (2013). Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics. Nat. Meth. 10, 570–576.

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Facility Head Marc Moniatte

Scientific Collaborators Diego Chiappe Florence Armand Adrian Schmid Research Assistants Romain Hamelin Jonathan Paz-Montoya Lisa Pilet Adminstrative Assistant Maureen Hersperger

... contributes also to collaborative based services requiring heavy involvement of both parties such as:

• Lipid

Team Members

Contact Information Dr. Marc Moniatte AI 0149, Station 15 CH-1015, Lausanne +41 (0)21 693 17 53 marc.moniatte@epfl.ch

EPFL School of Life Sciences - 2014 Annual Report

Protein Crystallography - PCRYCF http://pcrycf.epfl.ch

The Protein Crystallography Core Facility provides instrumentation and expertise at every stage of the structure determination process for noncrystallography groups who are interested in solving the structures of their favorite macromolecule. Expertise and advice include consultation on protein purification, crystallization, and crystal optimization, as well as assistance with X-ray crystal screening, data collection, data processing and structure determination and analysis. X-ray crystallography is the primary method for determining three-dimensional structures of biological macromolecules, and is therefore an essential tool, which should be available to a broad range of researchers. Nowadays, it is possible for a noncrystallographer to access this technology thanks to automation and a variety of commercially available kits as well as to the friendlier and more intuitive programs that have been developed in recent years. With personalized advice, training, and follow-up, users are in the optimal environment to manage their crystallization screens, and to solve, refine and analyze the structures of their favorite proteins.

Services and Technologies • Advice on larger-scale protein expression and purification, if required. • Set-up of crystallization screens using commercial and facility-made conditions. • Optimization of crystals. • Data collection of quality crystals at facility x-ray source and synchrotrons. • Data processing using popular packages such as XDS and Mosflm. • Structure determination using molecular replacement and MAD techniques. • Structure refinement, fitting and analysis using ccp4i and Phenix software.

Team Members Facility Head Florence Pojer

Technician Aline Reynaud Administrative Assistant Manuelle Mary Contact Information Florence Pojer EPFL SV PTCRYP SV3827 Station 19 CH-1015 Lausanne Tel: +41 (0) 21 693 1438 pcrycf-platform@epfl.ch

• Deposition of structures in the protein database. • Preparation of images for publication using PyMol software.

Selected Publications » » » »

Neres J et al. (2014). 2-Carboxyquinoxalines Kill Mycobacterium tuberculosis through Noncovalent Inhibition of DprE1. ACS Chem Biol. Dec 9 Chen S et al. (2014). Peptide Ligands Stabilized by Small Molecules. Angew Chem Int Ed Engl. 53(6):1602-6 Hartkoorn RC et al. (2014) Pyridomycin bridges the NADH- and substrate-binding pockets of the enoyl reductase InhA. Nat Chem Biol. 10(2):96-8 Haruki H et al. (2013). Tetrahydrobiopterin biosynthesis as an off target of sulfa drugs. Science. 340(6135):987-91

167

Core Facilities & Technology Platforms

Introduction

EPFL School of Life Sciences - 2014 Annual Report

Protein Expression - PECF http://pecf.epfl.ch

Introduction

The objective of the PECF is to provide recombinant proteins, rapidly and at low cost, to EPFL researchers. Both cultivated mammalian cells and E. coli are used as production hosts. One of our main activities is recombinant protein production by transient transfection of Chinese hamster ovary (CHO) or human embryo kidney (HEK293) cells in suspension at volumetric scales from 5 mL to 10 L. We also produce proteins from existing recombinant cell lines developed by our clients. This may involve adapting the cell line to serum-free suspension culture. In this case, cultures at volumetric scales up to 10 L are used. For protein production in mammalian cells, we have a number of expression vectors available. We produce monoclonal antibodies by scale-up of existing hybridoma cell lines. When using E. coli as a host for protein production, the scales of operation range up to 20 L. For all the types of production mentioned, the PECF has non-instrumented bioreactors available for use. After production, we also provide services in protein recovery, mainly by affinity chromatography of proteins secreted from mammalian cells (antibodies and Fc- and his-tagged proteins) and GSTand his-tagged proteins produced in E. coli. For protein production in mammalian cells, we have a number of expression vectors available.

Services and Technologies • Large-scale transient transfection for recombinant protein in mammalian cells • Scale-up of existing cell lines for recombinant protein production • Scale-up of existing hybridoma cell lines for monoclonal antibody production • Recombinant protein production in E. coli • Affinity protein purification • Provision of vectors for protein production in mammalian cells

Selected Publications » Shen, X., Hacker, D.L., Baldi, L., and Wurm, F.M. (2014). Virus-free transient protein production in Sf9 cells. J. Biotechnol. 171:61-70. » Kilpinen, H., Waszak, S.M., Gschwind, A.R., Raghav, S.K., Witwicki, R.M., Orioli, A., Migliavacca, E., Wiederkehr, M., Gutierrez-Arcelus, M., Panousis, N.I., Yurovsky, A., Lappalainen, T., Romano-Palumbo, L., Planchon, A., Bielser, D., Bryois, J., Padioleau, I., Udin, G., Thurnheer, S., Hacker, D., Core, L.J., Lis, J.T., Hernandez, N., Reymond, A., Deplancke, B., and Dermitzakis, E.T. (2013). Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription. Science 342(6159):744-747. » Hacker, D.L., Kiseljak, D., Rajendra, Y., Thurnheer, S., Baldi, L., Wurm, F.M. (2013). Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells. Protein Expr. Purif. 92(1):67-76. » Alattia, J.R., Matasci, M., Dimitrov, M., Aeschbach, L., Balasubramanian, S., Hacker, D.L., Wurm, F.M., Fraering, P.C. 2013. Highly efficient production of the Alzheimer’s γ-secretase integral membrane protease complex by a multi-gene stable integration approach. Biotechnol. Bioeng. 110(7):1995-2005.

168

Team Members Facility Head David Hacker

Technicians Laurence Durrer Soraya Quinche Contact Information David Hacker EPFL SV PECF Station 6, CH J2 506 CH-1015 Lausanne Tel: +41 (0)21 639 6142 david.hacker@epfl.ch

EPFL School of Life Sciences - 2014 Annual Report

Centre of PhenoGenomics - Transgenic - CPG-TCF http://tcf.epfl.ch/

Genetic manipulation of rodents through the generation of transgenic animals is a procedure of primary importance for biomedical research, either to address fundamental questions or to develop preclinical models of human diseases. We offer a centralized resource with various technologies for the generation of transgenic animals including pronuclear injection of DNA, lentiviral vector injection in the mouse oocyte and embryonic stem cells injection into the blastocyst. We are also a general support in both vector design and lentiviral vector production and titration, as our expertise in lentiviral vectors has become of general interest for many other applications than transgenesis. An important variable that affects the results of mouse studies is the sanitary status of the animals. Taking advantage of our expertise in embryo manipulation we also propose the sanitary cleaning of mouse transgenic lines by embryo transfer as a routine service. This procedure allows cleaning and hosting of a wide range of mouse lines in the SPF (Specific Pathogen Free) area of the EPFL animal facility.

Services and Technologies • Pronuclear injection: plasmids and BACs. • Lentiviral vector mediated transgenesis. • ES cells microinjection into blastocysts. • Lentiviral vectors production/titration. • Vectorology. • Cleaning of established mouse lines by embryo transfer. • Cryopreservation of mouse lines by sperm freezing. • In vitro fertilization assay (IVF).

Team Members Facility Head Isabelle Barde

Collaborators Dalil Ait-Bara Michelle Blom Sandra Offner Sonia Verp Contact Information: Isabelle Barde EPFL SV CPG-GE SV 1836, Station 19 CH-1015 Lausanne +41 (0)21 693 17 02 isabelle.barde@epfl.ch

For long-term preservation of mouse line of particular interest, we now propose cryopreservation by sperm freezing and recovery by in vitro fertilization (IVF).

Selected Publications » Lonfat, N., Montavon, T., Darbellay, F., Gitto, S., Duboule, D. (2014) Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci. Science;346(6212):1004-6. » Delpretti, S., Montavon, T., Leleu, M., Joye, E., Tzika, A., Milinkovitch, M., Duboule, D. (2013) Multiple enhancers regulate Hoxd genes and the Hotdog LncRNA during cecum budding. Cell Rep.;5(1):137-50. » Andrey, G., Montavon, T., Mascrez, B., Gonzalez, F., Noordermeer, D., Leleu, M., Trono, D., Spitz, F., Duboule, D. (2013) A switch between topological domains underlies HoxD genes collinearity in mouse limbs. Science.;340(6137):1234167. » Gubelmann, C., Waszak, S.M., Isakova, A., Holcombe, W., Hens, K., Iagovitina, A., Feuz, J.D., Raghav, S.K., Simicevic, J., Deplancke, B. (2013) A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks. Mol Syst Biol.;9:682.

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Core Facilities & Technology Platforms

Introduction

EPFL School of Life Sciences - 2014 Annual Report

Center of PhenoGenomics – Phenotyping Unit - CPG-UDP

Introduction

The development of genetic tools for the manipulation of the mouse genome has led to the creation of numerous and sophisticated mouse models. The in-depth characterization of the phenotype of these mouse lines is crucial to decipher the roles of the genes of interest. The Phenotyping Unit of the CPG is composed of highly interactive service platforms including clinical chemistry laboratory, metabolic and functional exploration platform, behavior and cognition exploration platform. The UDP provides a range of state-of-the-art equipment to enable cardio-metabolic, biochemical, oncologic and behavioural exploration of mouse models. We offer different types of support to the users of the platform, going from general support and training in protocols establishment to full completion of tests and analysis. We benefit from the scientific expertise of professors of the School of Life Sciences, such as Prof. Johan Auwerx and Prof. Carmen Sandi, both experts in their respective fields, namely cardio-metabolism and neurobiology.

Services and Technologies

Team Members

A series of tests can be combined in a pipeline in order to answer questions related to a given topic such as neurodegenerative diseases or obesity or diabetes.

CPG – UDP Managers Roy Combe Raphaël Doenlen

The in-vivo imaging platform allows monitoring of disease progress in oncology by the assessment of tumor development, progression, metastasis, and response to therapy in models of cancer. Imaging modalities include high resolution CT scan, ultrasound and optical imaging (invivo bioluminescence or fluorescence). They improve cancer research by allowing longitudinal and non-invasive assessment.

Collaborators Arnaud Bichat Cristina Cartoni Sébastien Lamy Adeline Langla Elodie Schneider Céline Waldvogel Camille Aebischer Sybil Bron Cindy Bula Christine Pehm

We offer tests in the different scientific fields mentioned in the opposite figure.

The UDP is part of the animal facility barrier unit, and encompasses a working area constituted of housing, testing and analysis rooms. The mouse models are housed in individual ventilated cages and maintained at a conventional sanitary status. The UDP equipment has been chosen to ensure a high level of flexibility for the tests that can be performed. Additionally, most of experiments can be run by fully programmable and automated interfaces and thus the impact of experimental interventions by the researcher over the experimental period is reduced. Selected Publications » Trachet, B., Fraga-Silva, R.A., Piersigilli, A., Tedgui, A., Sordet-Dessimoz, J., Astolfo, A., Van der Donckt, C., Modregger, P., Stampanoni, M.F., Segers, P. and Stergiopulos, N. (2015). Dissecting abdominal aortic aneurysm in Ang II-infused mice: suprarenal branch ruptures and apparent luminal dilatation. Cardiovasc Res. 105(2):213-22. » Boutant, M., Joffraud, M., Kulkarni, S.S., García-Casarrubios, E., García-Roves, P.M., Ratajczak, J., Fernández-Marcos, P.J., Valverde A.M., Serrano M., and Cantó C. (2014). SIRT1 enhances glucose tolerance by potentiating brown adipose tissue function. Mol. Metab. 4(2):118-31. » Doenlen, R., Cettour-Rose, P., Bichat, A., Cartoni, C., Lamy, S., Langla, A., Varet, M. and Warot, X., (2013). Impact of Fenbendazole on neurobiological and metabolic functions in mice. FELASA meeting. June 10-13. » Cantó, C., Houtkooper, R.H., Pirinen, E., Youn, D.Y., Oosterveer, M.H., Cen, Y., Fernandez-Marcos, P.J., Yamamoto, H., Andreux, P.A., Cettour-Rose, P., Gademann, K., Rinsch, C., Schoonjans, K,. Sauve, A.A. and Auwerx, J. (2012). The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cell Metabolism. 15(6):838-847.

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Facility Head Xavier Warot

Contact Information Xavier Warot CPG head Tel: +41 (0) 21 693 1869 xavier.warot@epfl.ch Raphaël Doenlen UDP manager Tel: +41 (0) 21 693 0953 raphael.doenlen@epfl.ch

Core Facilities & Technology Platforms

EPFL School of Life Sciences - 2014 Annual Report

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EPFL School of Life Sciences - 2014 Annual Report

11th edition 2014/2015 Produced and edited by the EPFL School of Life Sciences Printed at the EPFL “Atelier de Reprographie” Image - Christopher Tremblay (Prof. Melody Swartz Lab) see p.2

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Editor: Alice Emery-Goodman With many thanks to Bruno Liardon (photographs & cover design), Roland Chabloz (Reprographie), and Harald Hirling for their help and support!