Scripps Research Institute View Institution's Website 12 articles published in JoVE Biology Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy Benjamin Basanta*1, Wenqian Chen*1, Daniel E. Pride1, Gabriel C. Lander1 1Department of Structural and Computational Biology, Scripps Research Here, we describe a protocol for applying a single monolayer of graphene to electron microscopy grids and how to prepare them for use in cryoEM structure determination. Chemistry Hierarchical and Programmable One-Pot Oligosaccharide Synthesis Cheng-Wei Cheng1, Yixuan Zhou1, Wen-Harn Pan2, Supriya Dey3, Chung-Yi Wu1, Wen-Lian Hsu4, Chi-Huey Wong1,3 1Genomics Research Center, Academia Sinica, 2Institute of Biomedical Sciences, Academia Sinica, 3Department of Chemistry, Scripps Research Institute, 4Institute of Information Science, Academia Sinica This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination experiments and one-pot glycosylation of SSEA-4. Neuroscience Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity Jesse Zhan1, Ruchi Komal1, William T. Keenan2, Samer Hattar1, Diego C. Fernandez1 1Section on Light and Circadian Rhythms (SLCR), National Institute of Mental Health, 2Howard Hughes Medical Institute, Department of Neuroscience, Scripps Research Institute Here we describe two non-invasive methods to chronically control neuronal activity using chemogenetics in mice. Eye-drops were used to deliver clozapine-N-oxide (CNO) daily. We also describe two methods for prolonged administration of CNO in drinking water. These strategies for chronic neuronal control require minimal intervention reducing animals’ stress. Genetics Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes Jingxin Wang1, John Hammond2, Kristen A. Johnson1 1Calibr, The Scripps Research Institute, 2Department of Integrative Structural and Computational Biology, The Scripps Research Institute Detailed protocols for both in vitro and in-cell selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article. Immunology and Infection Antigenic Liposomes for Generation of Disease-specific Antibodies Kyle J. Bednar*1, Lakeya Hardy*2,3, Johanna Smeekens*3,4, Dharmendra Raghuwanshi5, Shiteng Duan6, Mike D. Kulis3,4, Matthew S. Macauley5,7 1Janssen R&D, 2Department of Microbiology and Immunology, University of North Carolina, 3UNC Food Allergy Initiative, University of North Carolina, 4Department of Pediatrics, University of North Carolina, 5Department of Chemistry, University of Alberta, 6Department of Molecular Medicine, Scripps Research Institute, 7Department of Medical Microbiology and Immunology, University of Alberta Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models. Biology Generation of Mice Derived from Induced Pluripotent Stem Cells Michael J. Boland1, Jennifer L. Hazen1, Kristopher L. Nazor1, Alberto R. Rodriguez2, Greg Martin2, Sergey Kupriyanov2, Kristin K. Baldwin1 1Dorris Neuroscience Center & Department of Cell Biology, The Scripps Research Institute, 2Mouse Genetics Core Facility, The Scripps Research Institute Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1. Biology Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts Urszula Polak1,2, Calley Hirsch1, Sherman Ku3, Joel Gottesfeld3, Sharon Y.R. Dent1, Marek Napierala1 1Department of Molecular Carcinogenesis and Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center, 2Department of Cell Biology, Poznan University of Medical Sciences, 3Department of Molecular Biology, The Scripps Research Institute We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody. Medicine Teratoma Generation in the Testis Capsule Suzanne E. Peterson1, Ha T. Tran1, Ibon Garitaonandia1, Sangyoon Han1, Kyle S. Nickey1, Trevor Leonardo2, Louise C. Laurent3, Jeanne F. Loring1 1Department of Chemical Physiology, Scripps Research Institute, 2Department of Chemical Physiology, Scripps Research Institute, 3Department of Reproductive Medicine, University of California Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs. Biology Flow Cytometry Purification of Mouse Meiotic Cells Irina V. Getun1, Bivian Torres2, Philippe R.J. Bois1 1Genome Plasticity Laboratory, Department of Cancer Biology, The Scripps Research Institute, 2Flow Cytometry Core, The Scripps Research Institute An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions. Biology Crystallization of Membrane Proteins in Lipidic Mesophases Wei Liu1, Vadim Cherezov1 1Molecular Biology, The Scripps Research Institute The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals. Biology Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates Gregory M. Solis1,2, Michael Petrascheck1,2 1Department of Chemical Physiology, The Scripps Research Institute, 2Department of Molecular and Experimental Medicine, The Scripps Research Institute In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates. Biology Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM) James Lim1, Gaudenz Danuser1 1Department of Cell Biology, Scripps Institute Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).