National Research Council of Italy 12 articles published in JoVE Bioengineering In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing Alessandro Migliara1, Martino Alfredo Cappelluti1, Francesca Giannese2, Sara Valsoni1, Alberto Coglot1, Ivan Merelli1,3, Davide Cittaro2, Angelo Lombardo1,4 1San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 2Center for Omics Sciences, IRCCS San Raffaele Institute, 3Institute for Biomedical Technologies, National Research Council, 4Vita-Salute San Raffaele University Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings. Immunology and Infection A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice Sonia Simonetti*1,2, Ambra Natalini*1,2, Giovanna Peruzzi3, Alfredo Nicosia4, Antonella Folgori5, Stefania Capone5, Angela Santoni2,6, Francesca Di Rosa1 1Institute of Molecular Biology and Pathology, National Research Council of Italy (CNR), 2Department of Molecular Medicine, University of Rome “Sapienza”, 3Center for Life Nano Science, Istituto Italiano di Tecnologia, 4Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 5Reithera Srl, 6IRCCS, Neuromed Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice. Engineering Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering Rajeev Ranjan1, Maurizio Indolfi1, Maria Antonietta Ferrara1, Luigi Sirleto1 1National Research Council, Institute for Microelectronics and Microsystems, Naples Unit, Italy In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO). Developmental Biology Methods to Test Endocrine Disruption in Drosophila melanogaster Tiziana Francesca Bovier1, Daniela Cavaliere1, Michele Colombo1, Gianfranco Peluso2, Ennio Giordano3, Filomena Anna Digilio1,2 1Institute of Biosciences and BioResources, National Research Council of Italy (CNR), 2Institute of Research on Terrestrial Ecosystems (IRET), National Research Council of Italy (CNR), 3Department of Biology, UniNa "Federico II" Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly. Biology In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells Francesco Castagnetti1,2, Elisabetta Fiacco3, Carol Imbriano4, Lucia Latella1,2 1Department of Medicine, Institute of Translational Pharmacology, Italian National Research Council, 2Epigenetics and Regenerative Medicine, IRCCS Fondazione Santa Lucia, 3Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), 4Department of Life Sciences, University of Modena and Reggio Emilia Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice. Medicine Ultrasound-based Pulse Wave Velocity Evaluation in Mice Nicole Di Lascio1,2, Claudia Kusmic2, Francesco Stea2,3, Francesco Faita2 1 Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites. Biology Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells Loredana Moro1, Nicoletta Guaragnella1, Sergio Giannattasio1 1Institute of Biomembranes and Bioenergetics, National Research Council Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. Environment Protocols for Robust Herbicide Resistance Testing in Different Weed Species Silvia Panozzo1, Laura Scarabel1, Alberto Collavo1, Maurizio Sattin1 1Institute of Agro-environmental and Forest Biology (IBAF), National Research Council (CNR), Italy A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations. Neuroscience Laser Nanosurgery of Cerebellar Axons In Vivo Anna L. Allegra Mascaro1, Leonardo Sacconi1,2, Francesco Saverio Pavone1,2,3,4 1European Laboratory for Non-Linear Spectroscopy, University of Florence, 2National Institute of Optics, National Research Council, 3Department of Physics and Astronomy, University of Florence, 4International Center for Computational Neurophotonics (ICON Foundation) Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo. Neuroscience A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions Antonio G. Zippo1, Sara Nencini1, Gian Carlo Caramenti2, Maurizio Valente1, Riccardo Storchi3, Gabriele E.M. Biella1 1Institute of Molecular Bioimaging and Physiology (IBFM), Department of Biomedicine, National Research Council, 2Institute of Biomedical Technologies (ITB), Department of Biomedicine, National Research Council, 3Faculty of Life Sciences, University of Manchester To elucidate the complex transition from Local Field Potentials (LFPs) to spikes a suitable stimulator for light mechanical peripheral stimuli was built. As an application, the spiking activities recorded from somatosensory cortex were analyzed by a multi-objective optimization strategy. The results demonstrated that the proposed stimulator was able to deliver tactile stimuli with millisecond and millimeter precisions. Biology Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells Elisa Di Pasquale1,2, Belle Song1, Gianluigi Condorelli1 1Humanitas Clinical and Research Center, Italy, 2Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR) Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol. Bioengineering Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution Massimo Vassalli1, Michele Basso2, Francesco Difato3 1Institute of Biophysics, National Research Council of Italy, 2Dipartimento di Sistemi e Informatica, Università di Firenze, 3Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.
