EPFL Life Sciences Core Faclities 2011 by EPFL School of Life Sciences

EPFL School of Life Sciences - 2011 Annual Report

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 state-of-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. 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://unil.ch/dafl).

Core Facilities & Technology Platforms

The following pages describe the Life Sciences-related core facilities and technology platforms currently available at the EPFL School of Life Sciences.

CORE

EPFL School of Life Sciences - 2011 Annual Report

Bioelectron Microscopy - Bio-EM http://cime.epfl.ch/bio-em

Team Members Facility Head Graham Knott

Postdoctoral researchers Natalya Korogod Bohumil Maco Scientists Davide Demurtas Corrado Cali Technicians Marie Croisier Stéphanie Rosset

Introduction

The Bio Electron Microscopy Facility (BioEM) is located in the Faculty of Life Science (SV), and also in the Interdisciplinary Centre of Electron Microscopy (CIME). It provides both services and training to researchers at the EPFL who need to image their biological samples at high resolution. This begins with specific sample preparation techniques, carried out in the preparation laboratories, and then a range of different imaging approaches using either scanning or transmission electron microscopes. These machines are specially suited to biological samples and during 2011, the facility expanded its range of imaging technology with the acquisition of two new transmission electron microscopes; one for ambient temperature imaging, and the other for imaging frozen samples. Other additions during the past year also include the installation of a new high-pressure freezer for instantaneously freezing living material.

Services and Technologies • Transmission electron microscopy • Cryo transmission electron microscopy • Scanning electron microscopy • Focussed ion beam scanning electron microscopy • Correlated light and electron microscopy • Resin embedding • Semithin sectioning • Ultrathin sectioning • Serial sectioning • Cryosectioning and immunolabelling • Pre-embedding immuno labelling • Negative staining • Critical point drying • High pressure freezing • Plunge freezing • Low temperatureand, freeze substitution embedding

Selected Publications

Knott, Graham, Stéphanie Rosset, and Marco Cantoni. «Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue.» Journal of visualized experiments : JoVE , no. 53 (2011) Lucchi, A, K Smith, R Achanta, G Knott, and P Fua. «Supervoxel-Based Segmentation of Mitochondria in EM Image Stacks with Learned Shape Features.» IEEE transactions on medical imaging (2011) Straehle, C N, U Köthe, G Knott, and F A Hamprecht. «Carving: Scalable Interactive Segmentation of Neural Volume Electron Microscopy Images.» Med Image Comput Comput Assist Interv 14, no. Pt 1 (2011): 653-60. Kreshuk, Anna, Christoph N Straehle, Christoph Sommer, Ullrich Koethe, Marco Cantoni, Graham Knott, and Fred A Hamprecht. «Automated Detection and Segmentation of Synaptic Contacts in Nearly Isotropic Serial Electron Microscopy Images.» PloS one 6, no. 10 (2011). Cantoni, M. Genoud, C., Hébert, C., Knott, GW. (2010). Large volume, isotropic, 3D imaging of cell structure on the nanometer scale. Microscopy and Analysis 24 (4).

Contact Information: Graham Knott Station 19, EPFL CH-1015 Lausanne Tel: +41 (0) 21 693 1862 graham.knott@epfl.ch

EPFL School of Life Sciences - 2011 Annual Report

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

Team Members Facility Head Arne Seitz

Collaborators José Artacho Olivier Burri Mathias Fournier Romain Guiet Thierry Laroche

The Bioimaging and Optics platform (PT-BIOP) is located in the faculty of Life Science (SV) at the Ecole Polytechnique Fédérale de Lausanne (EPFL) and is part of a network of core facilities at the institute. The general idea of the platform is to provide state of the art light microscopes and even more importantly, expertise to solve challenging (biological) questions with modern light-microscopy. Currently 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 are available in the facility. Scientists who want to make use of the available equipment are trained by the PT-BIOP staff so that they can use the instruments independently or under the supervision of the staff. Additionally there is a strong competence and necessary computer power to perform image processing. The idea is to link the image analysis with the image acquisition as early as possible as only this approach guarantees optimal scientific results. The microscopes and the image analysis capabilities can be used by scientists of the faculty and the EPFL but are also available to scientist coming from outside the EPFL.

Services and Technologies • Wide-field transmission and fluorescent microscopes • Life cell imaging microscopes

Selected Publications

Bélanger M, Yang J, Petit JM, Laroche T, Magistretti PJ, Allaman I. Role of the glyoxalase system in astrocyte-mediated neuroprotection. J Neurosci. (2011) 31(50):18338-52. Terjung, S., Walter, T., Seitz, A., Neumann, B., Pepperkok, R., and Ellenberg, J. (2010) High-throughput microscopy using live mammalian cells, (2010) Cold Spring Harbor Protocols, pdb top84. Maurel, D., Banala, S., Laroche, T., and Johnsson, K. (2010) Photoactivatable and photoconvertible fluorescent probes for protein labeling, ACS Chem Biol 5, 507-516. Kobel, S., Limacher, M., Gobaa, S., Laroche, T., and Lutolf, M. P. (2009) Micropatterning of hydrogels by soft embossing, Langmuir 25, 8774-8779. Lefort S., Tomm C., Floyd Sarria J.C., Petersen C.C. (2009) The excitatory neuronal network of the C2 barrel column in mouse primary somstosensory cortex, Neuron 61, 301-316. Emmenlauer, M., Ronneberger, O., Ponti, A., Schwarb, P., Griffa, A., Filippi, A., Nitschke, R., Driever, W., and Burkhardt, H. (2009) XuvTools: free, fast and reliable stitching of large 3D datasets, J Microsc 233, 42-60.

Contact Information: Arne Seitz AI 0240 Station 19, EPFL CH-1015 Lausanne Tel: +41 (0) 21 693 9618 Fax: +41 (0) 21 693 9585 arne.seitz@epfl.ch

Core Facilities & Technology Platforms

Introduction

• Single and multiple-beam confocal microscopes • 2P microscope • High resolution and super resolution microscope (will be available in 2012) • Image Processing tools (commercially available and/or custom built)

EPFL School of Life Sciences - 2011 Annual Report

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

Team Members Facility Head Jacques Rougemont Post doctoral Solenne Carat Fabrice David Julia di Iulio Gregory Lefebvre Marion Leleu Scientific assistants Julien Delafontaine Yohan Jarosz Bara’ah Khubieh Frederick Ross Lucas Sinclair Administrative Assistant Sophie Barret

Introduction and Services and Technologies

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 development of data management pipelines for new high-throughput technologies (e.g. high-density arrays, high-throughput sequencers) and for the statistical planning in complex experimental designs. It also helps researchers in the areas of 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, and in particular the Vital-IT high performance computing center.

Selected Publications

Ayyanan, A., Laribi, O., Schuepbach-Mallepell, S., Schrick, C., Gutierrez, M., Tanos, T., Lefebvre, G., et al. (2011). Perinatal exposure to bisphenol a increases adult mammary gland progesterone response and cell number. Molecular Endocrinology, 25(11), 1915–1923. Huber, A., French, S. L., Tekotte, H., Yerlikaya, S., Stahl, M., Perepelkina, M. P., Tyers, M., et al. (2011). Sch9 regulates ribosome biogenesis via Stb3, Dot6 and Tod6 and the histone deacetylase complex RPD3L. The EMBO journal, 30(15), 3052–3064. Rey, G., Cesbron, F., Rougemont, J., Reinke, H., Brunner, M., & Naef, F. (2011). Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver. PLoS Biology, 9(2), e1000595. Leleu, M., Lefebvre, G., & Rougemont, J. (2010). Processing and analyzing ChIP-seq data: from short reads to regulatory interactions. Briefings In Functional Genomics, 9(5-6), 466–476. Preti, M., Ribeyre, C., Pascali, C., Bosio, M. C., Cortelazzi, B., Rougemont, J., Guarnera, E., et al. (2010). The telomere-binding protein Tbf1 demarcates snoRNA gene promoters in Saccharomyces cerevisiae. Molecular Cell, 38(4), 614–620. Rowe, H. M., Jakobsson, J., Mesnard, D., Rougemont, J., Reynard, S., Aktas, T., Maillard, P. V., et al. (2010). KAP1 controls endogenous retroviruses in embryonic stem cells. Nature, 463(7278), 237–240.

Contact Information: Dr. Jacques Rougemont Station 15, CH-1015, Lausanne +41 (0)21 693 9573 jacques.rougemont@epfl.ch

Noordermeer, D., Leleu, M., Splinter, E., Rougemont, J., de Laat, W., & Duboule, D. (2011). The dynamic architecture of Hox gene clusters. Science, 334(6053), 222–225. Truman, R. W., Singh, P., Sharma, R., Busso, P., Rougemont, J., Paniz-Mondolfi, A., Kapopoulou, A., et al. (2011). Probable zoonotic leprosy in the southern United States. The New England Journal of Medicine, 364(17), 1626–1633.

EPFL for all material published in this report info.sv@epfl.ch

EPFL School of Life Sciences - 2011 Annual Report

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

Team Members Facility Head Gerardo Turcatti

Scientists Damiano Banfi Marc Chambon Ruud van Deursen Assistants Nathalie Ballanfat Manuel Bueno Miquel Busquets Julien Mena

The BSF provides access to EPFL, NCCR-Chemical Biology and SystemsX.ch researchers to the infrastructure, expertise and collections of molecules required for performing medium to high throughput molecular screening assays. In the frame of the NCCR-Chemical Biology, the BSF leads the project ACCESS with the main mission to become the platform for Academic Chemical Screens in Switzerland. In addition, the BSF is pursuing an innovative and focused research program with industrial partners in screening or drug discovery-linked areas. Most of the incoming projects are related to chemical biology, systems biology or disease-oriented research in particular in the areas of cancer, infectious diseases and neurobiology. Our multidisciplinary laboratory provides scientists with adequate screening instrumentation, state-of-the-art technologies and compounds collections for applications ranging from the probing of cellular pathways to the broad area of bioactive compounds research. We perform our automated screens in 96 and 384 well plates for the following two main categories of assays: • Screening of chemicals for a variety of biochemical target-based and cellular assays using large, chemically diverse collections • RNA interference (RNAi) cellular screens for probing gene function using collections of small interfering RNAs (siRNAs) targeting the human genome.

Services and Technologies

• Access to instrumentation dedicated to microplates and cell culture facilities • Assay development and validation for HTS • Assay automation and statistical validations • Pilot screening • Primary screening campaigns • Hits confirmation • Dose response assays • Secondary screens

• Compound storage and management of collections • Image processing for high content screening read-outs • Data management using in-house developed Laboratory Implementation Management System (LIMS). • Cheminformatics

Selected Publications

Takahashi-Umebayashi, M., Pineau, L., Hannich, T., Zumbuehl, A., Doval, D. A., Matile, S., Heinis, C., Turcatti, G., Loewith, R., Roux, A., lien, Reymond, L., Johnsson, K., and Riezman, H. (2011) Chemical Biology Approaches to Membrane Homeostasis and Function, CHIMIA International Journal for Chemistry 65, 849-852. Magnet, S., Hartkoorn, R. C., Székely, R., Pató, J., Triccas, J. A., Schneider, P., Szántai-Kis, C., Orfi, L., Chambon, M., Banfi, D., Bueno, M., Turcatti, G., Kéri, G., and Cole, S. T. (2010) Leads for antitubercular compounds from kinase inhibitor library screens, Tuberculosis 90, 354-360. Kobel, S., Valero, A., Latt, J., Renaud, P., and Lutolf, M. (2010) Optimization of microfluidic single cell trapping for long-term on-chip culture, Lab on a Chip 10, 857-863. Gormley, N.; Boutell, J., Turcatti, G.,Barnes, G. (2010). Preparation of nucleic acid templates for solid phase amplification. Patent number: US2010041561. Ouertatani-Sakouhi, H., El-Turk, F., Fauvet, B., Cho, M.-K., Pinar Karpinar, D., Le Roy, D., Dewor, M., Roger, T., Bernhagen, J., Calandra, T., Zweckstetter, M., and Lashuel, H. A. (2010) Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action, Journal of Biological Chemistry 285, 26581-26598.

Contact Information: Gerardo Turcatti, MER Station 15 EPFL CH-1015 Lausanne Switzerland Tel: +41-(0)21 693 9666 gerardo.turcatti@epfl.ch

Core Facilities & Technology Platforms

Introduction

CORE

EPFL School of Life Sciences - 2011 Annual Report

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

Team Members Facility Head Miguel Garcia

Collaborators Gonzalo Tapia Sintia Winkler Administrative Assistant Ursula Winter

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. Sorting allows us to capture and collect cells of interest for further analysis. Our mission is to provide comprehensive flow cytometric analysis and sorting including instrumentation, technical and professional assistance, training.

Services and Technologies

The Flow Cytometry Core Facility from EPFL is equipped with five self-service cytometers. For sorting, the facility has two high-speed sorters from BD (BD FACSAria II SORP & FACSVantage SE). The Facility also operates an automated immunomagnetic bead cell separator from Miltenyi Biotec MACS® Technology. The LSRII (Becton Dickinson) is a 5 lasers benchtop analyser capable of 18 colour, forward and side scatter analysis, equipped with a PC and DIVA digital acquisition software system. The Accuri C6 is equipped with 2 lasers and 4 active detectors to allow maximum flexibility for easy experimental design. This machine is also equipped with a plate reader (CSampler) The Cyan ADP (Beckman Coulter) is a 3-laser benchtop analyser capable of 9 colour, forward and side scatter analysis, equipped with a PC and Summit digital acquisition software system.

quick and quantitative analysis of red & green expression, apoptosis, cell viability, cell count, and much more. The AutoMACS Pro is a fully automated bench-top sorter that can be used to perform sterile bulk sorts. Designed for ultra high-speed positive selection as well as depletion, the AutoMACS Pro can isolate virtually any cell type. The FACSVantage DIVA (BD) is a 3-laser sorter capable of 8 colour, forward and side scatter analysis. It is equipped with DIVA digital acquisition software system. The FACSAria (BD) is a 5 laser high-speed sorter capable of 18 colour, forward and side scatter analysis. It is equipped with DIVA digital acquisition software system and ACDU. Services : • Cell sorting • User training • Help with acquisition and data analysis • Experiment design & manuscript • Advice on cell preparation • Interpretation of results • Consulting

Contact Information: Miguel Garcia AI 0147 Sation 15 EPFL CH – 1015 Lausanne Tel: +41 21 693 0901 miguel.garcia@epfl.ch

The TaliTM Image Cytometer is a 3-channel (bright field, green & red fluorescence) benchtop assay platform giving a

EPFL School of Life Sciences - 2011 Annual Report

Histology -

HCF & Comparative Pathology

http://hcf.epfl.ch

Team Members Facility Head Jessica Sordet-Dessimoz Collaborators Gian-Filippo Mancini Nathalie Müller Agnès Hautier Veterinary pathologist Fabio Aloisio Administrative Assistant Ursula Winter

Introduction

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 nothing without the expertise of a specialist in veterinarian pathology who has been hired in 2011 to help researchers analyzing their slides.

Pathology service Pathology support is provided by professionals that underwent formal postgraduate training in veterinary anatomic pathology officially acknowledged by board certification of specialty. These professionals are trained to interpret morphologic changes within organs and tissues processed through the variety of histology techniques. Appropriate interpretation of tissue changes implies proper recognition of tissue abnormalities and pathologic processes of diseases that manifest as morphologic changes observable in histological preparations.

Services and Technologies

The service provides the following activities:

On the other hand technicians of the facility perform work for researchers: • Tissue processing to frozen, paraffin or resin sections • Histological stains like the standard Hematoxyline and eosin and routine stains like Masson’s trichrome or cresyl violet among others. The standard stains are running on the Prisma automate from Sakura • Setup and optimization of immunohistochemistry and immunofluorescence protocols • Detection of mRNA and miRNA using cold probes on the Discovery xT automate from Roche-Ventana.

• Consulting, at the study design level for issues related to pathology investigation • Phenotyping, whole body or organ targeted for genetically engineered animals • Analysis (morphology), of histological preparations

Core Facilities & Technology Platforms

On one hand, 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. Furthermore a large panel of secondary antibodies are titrated and provided to the researchers by the service.

• Support, in reporting pathology data for manuscript preparation and grant application • Diagnostics. Post mortem examination of diseased animals within the colony.

Contact Information: Jessica Sordet-Dessimoz EPFL SV PTH AI 0342 Station 19 1015 Lausanne +41 (0)21 693 0962 info.hcf@epfl.ch

CORE

EPFL School of Life Sciences - 2011 Annual Report

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

Team Members Facility Head Marc Moniatte

Collaborators Diego Chiappe Florence Armand Adrian Schmid Research Assistants Romain Hamelin Jonathan Paz-Montoya Administrative Assistant Sophie Barret

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. Today it is possible to get quantitative information about thousands of proteins in one experiment allowing researchers to begin to think more globally. But there is still room for very detailed studies on single proteins especially those modified by post-translational modifications.

Contributes also to collaborative based services requiring heavy involvement of both parties like: • Accurate protein quantification by SRM-MRM. • Localization and eventually quantification of PTM’s other than phosphorylation. • Lipid mixtures profiling.

The EPFL Proteomics Core Facility is a technological platform that has been created to address these needs and help researchers in using these techniques.

Selected Publications

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 ion traps, and 2 QQQ LC-ESI-MS/MS and 1 MALDI-TOF/TOF instruments. 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, Proteome 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.

Maintains tight collaboration with other proteomics facilities (UNIL-PAF, UNIGE-PCF, UNIBE) within a network called Repp-SO and with computer science and bioinformatics research centers (Vital-IT, SIB, etc..). Dastidar EG, Dayer G, Holland ZM, Dorin-Semblat D, Claes A, Chêne A, Sharma A, Hamelin R, Moniatte M, Lopez-Rubio J-J, Scherf A, Doerig C. (2012) Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway. BMC Biology Jan;10(1):5. Sicard A, Semblat J, Doerig C, Hamelin R, Sicard, A., Semblat, J. P., Doerig, C., Hamelin, R., Moniatte, M., Dorin-Semblat, D., Spicer, J. A., Srivastava, A., Retzlaff, S., Heussler, V., and Waters, A. P. (2011) Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes, Cell Microbiol. Jun;13(6):836-845. Schmid, A. W., Condemi, E., Tuchscherer, G., Chiappe, D., Mutter, M., Vogel, H., Moniatte, M., and Tsybin, Y. O. (2011) Tissue transglutaminase mediated glutamine deamidation of beta-amyloid peptide increases peptide solubility, whereas enzymatic cross-linking and peptide fragmentation may serve as molecular triggers for rapid peptide aggregation, J. Biol. Chem. Apr 8;286(14):12172-88. Paleologou KE, Oueslati A, Shakked G, Rospigliosi CC, Kim H-Y, Lamberto GR, Fernandez CO, Schmid A, Chegini F, Gai WP, Chiappe D, Moniatte M, Schneider BL, Aebischer P, Eliezer D, Zweckstetter M, Masliah E, Lashuel HA. (2010) Phosphorylation at S87 Is Enhanced in Synucleinopathies, Inhibits {alpha}Synuclein Oligomerization, and Influences Synuclein-Membrane Interactions. J. Neurosci, Mar;30(9):3184–3198. With acknowledgements Kitagawa D, Flückiger I, Polanowska J, Keller D, Reboul J, Gönczy P. (2011) PP2A phosphatase acts upon SAS-5 to ensure centriole formation in C. elegans embryos. Dev. Cell. Apr 19; 20(4):550-62

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

• Protein separation by FPLC and HPLC.

EPFL School of Life Sciences - 2011 Annual Report

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

Team Members Facility Head Florence Pojer

Collaborator Larry Richman

Introduction

The Protein Crystallography Core Facility provides instrumentation and expertise at every stage of the structure determination process for non-crystallography 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 are provided. 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. Presently,it is possible for a non-crystallographer 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 proteins of choice.

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

Selected Publications

Mollwitz B., Brun E., Schmitt S., Pojer F., Bannwarth M.,Rothlisberger U., Schiltz M.; Johnsson K. (2012). Directed evolution of the suicide protein O6-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability Biochemistry. Biochemistry 51(5):986-94. Blasco B, Stenta M, Alonso-Sarduy L, Dietler G, Peraro MD, Cole ST, Pojer F.* (2011). Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis. Molecular Microbiology 82(1):251-64.

Contact Information: Florence Pojer SV 3533 Station 19 EPFL CH-1015 Lausanne Tel: +41 (0)21 693 1772 +41 (0)21 693 1839 florence.pojer@epfl.ch

Core Facilities & Technology Platforms

Services and Technologies

The Protein Crystallography Core Facility provides the EPFL community with: • 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 xray source and synchrotrons. • Data processing using popular packages such as XDS and Mosflm. • Structure determination using molecular replacement, MAD and SAD techniques. • Structure refinement, fitting and analysis using ccp4i and Phenix software.

CORE

EPFL School of Life Sciences - 2011 Annual Report

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

Team Members Facility Head David Hacker

Collaborator Sarah Thurnheer

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 15 L using orbitally shaken bioreactors. For transient protein production in mammalian cells, we have a number of expression vectors available. With the same technical approach, we are also capable of producing virus vectors such as adeno associated virus. 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. Cultures at volumetric scales up to 15 L are possible. Expression vectors based on piggybac transposon-mediated gene delivery are available to our clients. We produce monoclonal antibodies by scale-up of existing hybridoma cell lines. Serum-free suspension cultures based on mixing by orbital shaking can be used for a scale-up to 2 liters. When using E. coli as a host for protein production, the scales of operation range from 100 mL – 20 L. Induced protein production at low temperatures is feasible. We also provide services in protein recovery, mainly by affinity chromatography of antibodies and tagged proteins (Fc, 6X his, FLAG and GST) produced in either mammalian cells or E. coli.

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

Matasci M, Baldi L, Hacker DL, Wurm FM. 2011. The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity. Biotechnol Bioeng. 108(9):2141-50. Rajendra Y, Kiseljak D, Baldi L, Hacker DL, Wurm FM. 2011. A simple highyielding process for transient gene expression in CHO cells. J Biotechnol. 153(1-2):22-6. Tissot S, Oberbek A, Reclari M, Dreyer M, Hacker DL, Baldi L, Farhat M, Wurm FM. 2011. Efficient and reproducible mammalian cell bioprocesses without probes and controllers? N Biotechnol 28(4):382-90. Xie Q, Michel PO, Baldi L, Hacker DL, Zhang X, Wurm FM. 2011. TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension. Biotechnol Lett. 33(5):897-902. Wurm FM, Hacker D. 2011. First CHO genome. Nat Biotechnol 29(8):718-20. Oberbek A, Matasci M, Hacker DL, Wurm FM. 2011. Generation of stable, high-producing CHO cell lines by lentiviral vector-mediated gene transfer in serum-free suspension culture. Biotechnol Bioeng. 108(3):600-10.

Contact Information: David Hacker Station 6 EPFL CH J2 496 CH-1015 Lausanne Tel: +41 (0)21 693 6142 david.hacker@epfl.ch

EPFL School of Life Sciences - 2011 Annual Report

Transgenic - TCF

http://jahia-prod.epfl.ch/cms/site/tcf

Team Members Facility Head Isabelle Barde

Collaborators Blom Michelle Guichard Sabrina Offner Sandra Eloise Verp Sonia

Introduction

Services and Technologies

We offer a centralized resource and state-of-the-art technology for the generation of transgenic animals. We can perform direct pronuclear injection of DNA in the mouse oocyte, which has been the standard method of trangenesis for more than three decades.

Selected Publications

As an attractive alternative, we are one of the very few platforms that provides a fast and efficient way to generate transgenic animals through the use of lentiviral vectors. Lentivector-mediated transgenesis is relatively easy to perform and leads to high percentages of provirus-positive animals. Moreover, a wide variety of lentiviral vectors have been developed that can all be used in transgenic animals, thus allowing for a broad range of genetic manipulations including externally controllable expression and knockdown, the latter offering an economically advantageous alternative to stable knockout. In addition to this primary service, we also offer 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 rederivation of mouse transgenic lines as a routine service. This procedure allows cleaning and hosting of a wide range of mouse lines in the SPF area of the EPFL animal house.

• Pronuclear injection: plasmids and BACs • Lentiviral vector mediated transgenesis • Vectorology • Lentiviral vectors production/titration • Rederivation by embryo transfer • Cryopreservation by sperm freezing • In progress: ES mediated transgenesis.

Barde I, Laurenti E, Verp S, Wiznerowicz M, Offner S, Viornery A, Galy A, Trumpp A, Trono D. (2011). Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease. Gene Ther. (11):1087-97. Robyr D, Friedli M, Gehrig C, Arcangeli M, Marin M, Guipponi M, Farinelli L, Barde I, Verp S, Trono D, Antonarakis SE.(2011). Chromosome conformation capture uncovers potential genome-wide interactions between human conserved non-coding sequences. PLoS One. 6(3):e17634. Friedli M, Barde I, Arcangeli M, Verp S, Quazzola A, Zakany J, Lin-Marq N, Robyr D, Attanasio C, Spitz F, Duboule D, Trono D, Antonarakis SE. (2010). A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the Olig1 and Olig2 locus. PLoS One.;5(12):e15741. Barde I, Salmon P, Trono D. (2010). Production and titration of lentiviral vectors. Curr Protoc Neurosci.;Chapter 4:Unit 4.21. Meyer K, Marquis J, Trüb J, Nlend Nlend R, Verp S, Ruepp MD, Imboden H, Barde I, Trono D, Schümperli D. (2009). Rescue of a severe mouse model for spinal muscular atrophy by U7 snRNA-mediated splicing modulation. Hum Mol Genet. 18(3):546-55.

Contact Information

Core Facilities & Technology Platforms

Genetic manipulation of rodents through the generation of transgenic animals is a procedure of paramount importance for biomedical research, either to address fundamental questions or to develop preclinical models of human diseases.

Isabelle Barde AI 3351 Station 19 EPFL CH-1015 Lausanne Tel: +41 (0)21 693 1702 isabelle.barde@epfl.ch

For long term preservation of a mouse line of particular interest, we now propose cryopreservation by sperm freezing via the JAX® Sperm Cryo Kit. The advantage of this technique is that it is standardized and requires only 2 competent male breeders.

CORE

EPFL School of Life Sciences - 2011 Annual Report

Phenotyping Unit - CPG-UDP /

Team Members CPG – Head Xavier Warot

CPG – UDP Managers Philippe Cettour-Rose Raphaël Doenlen Laboratory Assistants Arnaud Bichat Cristina Cartoni Sébastien Lamy Adeline Langla Marion Varet Animal Care Takers Christine Pehm

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 gene of interest. The clinical phenotyping unit of the Center of PhenoGenomics 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-ofthe-art equipment to enable cardio-metabolic, biochemical 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 for doing so from the scientific expertise of Prof. Johan Auwerx and Prof. Carmen Sandi, both authorities in their respective fields, namely cardio-metabolism and neurobiology. 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.

Services and Technologies

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

Selected Publications

Houtkooper RH, Argmann C, Houten SM, Cantó C, Jeninga EH, Andreux PA, Thomas C, Doenlen R, Schoonjans K, Auwerx J. (2011). The metabolic footprint of aging in mice. Sci Rep. 1:134. Marcaletti S, Thomas C, Feige JN. (2011). Exercise Performance Tests in Mice. Current Protocols in Mouse Biology. 1:141-154. Thomas C, Marcaletti S, Feige JN. (2011). Assessment of Spontaneous Locomotor and Running Activity in Mice. Current Protocols in Mouse Biology. 1:185198.

Contact Information:

Philippe Cettour-Rose : Cardio-metabolism Manager Tel: +41 (0)21 693 0984 philippe.cettour-rose@epfl.ch Raphael Doenlen: Neurobiology Manager Tel: +41 (0)21 693 0953 raphael.doenlen@epfl.ch Xavier Warot Tel: +41 (0)21 693 1869 xavier.warot@epfl.ch

Core Facilities & Technology Platforms

EPFL School of Life Sciences - 2011 Annual Report

EPFL for all material published in this report info.sv@epfl.ch