The Scripps Research Institute 22 articles published in JoVE Biochemistry Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon Sriram Aiyer1, Timothy S. Strutzenberg1, Marianne E. Bowman2, Joseph P. Noel2,3, Dmitry Lyumkis1,4,5 1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 3Department of Chemistry and Biochemistry, University of California San Diego, 4Graduate School of Biological Sciences, Section of Molecular Biology, University of California San Diego, 5Department of Integrative Structural and Computational Biology, The Scripps Research Institute The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface. Bioengineering Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery Kevin Eade1,2, Sarah Giles1,2, Sarah Harkins-Perry1,2, Martin Friedlander1,2 1Lowy Medical Research Institute, 2The Scripps Research Institute Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel). Biology Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice Andrea Galmozzi1,2, Bernard P. Kok1, Enrique Saez1 1Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA, 2Department of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, USA This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture. Environment RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points Shubham Rathore1, Jenni Hassert1, Courtney M. Clark-Hachtel2,3, Aaron Stahl1,4, Yoshinori Tomoyasu2, Elke K. Bushbeck1 1Department of Biological Sciences, University of Cincinnati, 2Department of Biology, Miami University, 3Department of Biology, University of North Carolina at Chapel Hill, 4Department of Neuroscience, The Scripps Research Institute RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior. Bioengineering Applications for Open Source Microplate-Compatible Illumination Panels Pierre Baillargeon1, Timothy P. Spicer1, Louis Scampavia1 1The Scripps Research Molecular Screening Center, Department of Molecular Medicine, Scripps Florida Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates. M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. In addition, it can assist with examining microplate quality or aid in the detection of errors. Biochemistry A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors Laszlo Radnai1,2, Rebecca F. Stremel1,2, James R. Sellers3, Gavin Rumbaugh2, Courtney A. Miller1,2 1Department of Molecular Medicine, The Scripps Research Institute, 2Department of Neuroscience, The Scripps Research Institute, 3Laboratory of Molecular Physiology, NHLBI, National Institutes of Health A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 µL per well. The platform should be applicable to virtually any ADP producing enzyme. Biology SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs Heike Fuhrmann-Stroissnigg*1, Fernando E. Santiago*1,2,3, Diego Grassi1, YuanYuan Ling1, Laura J. Niedernhofer1,2,3, Paul D. Robbins1,2,3 1Department of Molecular Medicine and the Center on Aging, The Scripps Research Institute, 2Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 3Institute on the Biology of Aging and Metabolism, University of Minnesota Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells. Biology Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands Tatiana Zyrianova1, Liana V. Basova1, Helen Makarenkova1 1Department of Molecular Medicine, The Scripps Research Institute The lacrimal gland (LG) has two cell types expressing α-smooth muscle actin (αSMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs. Immunology and Infection Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay Salome Murinello1, Stacey K. Moreno1, Matthew S. Macauley2, Susumu Sakimoto1, Peter D. Westenskow1,3, Martin Friedlander1 1Department of Cell and Molecular Biology, The Scripps Research Institute, 2Department of Chemical Physiology, The Scripps Research Institute, 3The Lowy Medical Research Institute Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting. Immunology and Infection A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins Ryan McBride1, James C. Paulson1, Robert P. de Vries1,2 1Department of Cell and Molecular Biology, Chemical Physiology and Microbial Science, The Scripps Research Institute, 2Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors. Biology Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses Catherine Cheng1, David S. Gokhin1, Roberta B. Nowak1, Velia M. Fowler1 1Department of Cell and Molecular Biology, The Scripps Research Institute Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals. Biology Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains Thomas Gaj*1, Jia Liu*1,2 1Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute, 2Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented. Developmental Biology Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells Peter Westenskow1,2, Zack Sedillo1,2, Ashley Barnett2, Martin Friedlander1,2 1Department of Cell and Molecular Biology, The Scripps Research Institute, 2Lowy Medical Research Institute Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells. Medicine Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension Peter D. Westenskow1,2, Toshihide Kurihara1, Stephen Bravo1, Daniel Feitelberg1, Zack A. Sedillo2, Edith Aguilar1, Martin Friedlander1,2 1Department of Cell and Molecular Biology, The Scripps Research Institute, 2Lowy Medical Research Institute Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration. Neuroscience Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis Sylvia Neumann1, George E. Campbell*1, Lukasz Szpankowski*2,3, Lawrence S.B. Goldstein2,4, Sandra E. Encalada1 1Department of Molecular and Experimental Medicine, Dorris Neuroscience Center, The Scripps Research Institute, 2Department of Cellular and Molecular Medicine, University of California San Diego, 3Department of Bioengineering, University of California San Diego, 4Department of Neurosciences, University of California San Diego School of Medicine Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins. Chemistry Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library Yu Gao1, Thomas Kodadek1 1Scripps Florida, The Scripps Research Institute Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs. Bioengineering Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology Xiaofeng Cui*1,2,3, Guifang Gao*2,4, Tomo Yonezawa5,6, Guohao Dai1 1Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 2Stemorgan Inc., 3Institute of Advanced Study, Technical University of Munich, 4Institute of Virology, School of Medicine, Wuhan University, 5Department of Molecular and Experimental Medicine, The Scripps Research Institute, 6Research Institute for Biomedical Sciences, Tokyo University of Science The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage. Biology One-channel Cell-attached Patch-clamp Recording Bruce A. Maki1, Kirstie A. Cummings2, Meaghan A. Paganelli1, Swetha E. Murthy3, Gabriela K. Popescu4 1Graduate Program in Neuroscience, SMBS, University at Buffalo, SUNY, 2Department of Biochemistry, SMBS, University at Buffalo, SUNY, 3Molecular and Cellular Neuroscience Department, The Scripps Research Institute, 4Department of Biochemistry and Graduate Program in Neuroscience, SMBS, University at Buffalo, SUNY Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes. Biology Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro Joseph Jablonski1, Mark Clementz1, Kevin Ryan2, Susana T. Valente1 1Department of Infectious Diseases, The Scripps Research Institute, 2Department of Chemistry, City College of New York RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro. Bioengineering Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons Komol Akhmedov1, Beena M. Kadakkuzha1, Sathyanarayanan V. Puthanveettil1 1Department of Neuroscience, The Scripps Research Institute, Florida Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry. Chemistry A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators Dejan Caglič1, Kristin M. Bompiani1, Michelle C. Krutein1, Petr Čapek1, Tobin J. Dickerson1,2 1Department of Chemistry, The Scripps Research Institute, 2Worm Institute of Research and Medicine (WIRM), The Scripps Research Institute The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity. Immunology and Infection Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells Benson Y.H. Cheng*1, Emilio Ortiz-Riaño*1, Juan Carlos de la Torre2, Luis Martínez-Sobrido1 1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Immunology and Microbial Sciences, The Scripps Research Institute Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.