Geisel School of Medicine at Dartmouth 10 articles published in JoVE Biochemistry A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors Kali A. Smolen1, Arminja N. Kettenbach1,2 1Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, 2Norris Cotton Cancer Center Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics. Neuroscience Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat Diana J. Wallin*1,2, Emily D. K. Sullivan*1,2, Elise M. Bragg1, Jibran Y. Khokhar2,4, Hanbing Lu2,3, Wilder T. Doucette1,2 1Dartmouth-Hitchcock Medical Center, 2Geisel School of Medicine at Dartmouth, 3National Institute on Drug Abuse, National Institutes of Health, 4University of Guelph This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine. Medicine Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome Jacob Vieira1, Tara Gallagher2, Hui-Yu Sui1, Sirus Jesudasen3, Katrine Whiteson2, George A. O'Toole4, Kurt Hanselmann5, Peggy S. Lai1 1Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, 2Department of Molecular Biology & Biochemistry, University of California, Irvine, 3Department of Medicine, Massachusetts General Hospital, 4Department of Microbiology & Immunology, Geisel School of Medicine at Dartmouth, 5Swiss i-research and teaching institute The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities. Immunology and Infection Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies Marta Rodriguez-Garcia1, Jared M. Fortier1, Fiona D. Barr1, Charles R. Wira1 1Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues. Neuroscience Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations Elise M. Bragg1, Farran Briggs1 1Physiology & Neurobiology, Geisel School of Medicine at Dartmouth This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses. Neuroscience Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis Francesca Gilli1, Darlene B. Royce1, Andrew R. Pachner1 1Department of Neurology, Geisel School of Medicine at Dartmouth An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice. Neuroscience Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection Catherine J. Fricano-Kugler1, Michael R. Williams1, Julia R. Salinaro1, Meijie Li1, Bryan Luikart1 1Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain. Neuroscience A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster Timothy P. Murphy1,2, Dan D. Luu1, Jessica A. DeSimone1,3, Thomas C. O'Brien1,4, Christopher J. Lally1,5, Jillian J. Lindblad1,6, Sarah M. Webster1 1Department of Biology, College of the Holy Cross, 2School of Medicine, Georgetown University, 3Department of Biochemistry, Giesel School of Medicine, Dartmouth College, 4School of Medicine, Tufts University, 5Transgenomic Inc., 6Department of Molecular, Cell and Cancer Biology, UMass Medical School In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal. Medicine Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer Melanie R. Rutkowski*1, Michael J. Allegrezza*1, Nikolaos Svoronos1, Amelia J. Tesone1, Tom L. Stephen1, Alfredo Perales-Puchalt1, Jenny Nguyen1, Paul J. Zhang2, Steven N. Fiering3, Julia Tchou4,5,6, Jose R. Conejo-Garcia1 1Tumor Microenvironment and Metastasis Program, Wistar Institute, 2Department of Pathology and Lab Medicine, Perelman School of Medicine, University of Pennsylvania, 3Department of Microbiology and Immunology and Department of Genetics, Geisel School of Medicine at Dartmouth, 4Division of Endocrine and Oncologic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, 5Rena Rowan Breast Center, Abramson Cancer Center, University of Pennsylvania, 6Center for Advanced Medicine, University of Pennsylvania Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer. Immunology and Infection Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii Leah M. Rommereim1, Miryam A. Hortua Triana1, Alejandra Falla1, Kiah L. Sanders1, Rebekah B. Guevara1, David J. Bzik1, Barbara A. Fox1 1Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth Here we report a method for using type I and type II Δku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.