Scripps Research Institute

11 articles published in JoVE

• 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 Wong^{1,3 1}Genomics 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. Fernandez^{1 1}Section 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. Johnson^{1 1}Calibr, 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. Kulis^3,4, Matthew S. Macauley^{5,7 1}Janssen 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. Baldwin^{1 1}Dorris 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 Polak^1,2, Calley Hirsch¹, Sherman Ku³, Joel Gottesfeld³, Sharon Y.R. Dent¹, Marek Napierala^{1 1}Department 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. Loring^{1 1}Department 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. Bois^{1 1}Genome 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 Cherezov^{1 1}Molecular 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. Solis^1,2, Michael Petrascheck^{1,2 1}Department 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 Danuser^{1 1}Department of Cell Biology, Scripps Institute Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).