University of California View Institution's Website 59 articles published in JoVE Immunology and Infection Experimental Model of Ligature-Induced Peri-Implantitis in Mice Davi Neto de Araújo Silva1, Maísa Casarin2, Sepehr Monajemzadeh1, Taciane Menezes da Silveira2, Jacob Lubben1, Beatriz Bezerra1, Flavia Q. Pirih1 1School of Dentistry, Section of Periodontics, University of California, 2School of Dentistry, Department of Periodontology, Federal University of Pelotas This is a report on an experimental model of ligature-induced peri-implantitis in mice. We describe all surgical steps, from pre- and post-operative management of the animals, extractions, implant placement, and ligature-induced peri-implantitis. Cancer Research Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures Katie B. Grausam*1, Joshua J. Breunig*1,2,3,4,5 1Board of Governor’s Regenerative Medicine Institute, Cedars-Sinai Medical Center, 2Center for Neural Sciences in Medicine, Cedars-Sinai Medical Center, 3Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, 5Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model. Biochemistry "Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome Akul Naik1, Sanjeeva Srivastava1,2, Arun P. Wiita1,3,4 1Department of Laboratory Medicine, University of California, San Francisco, 2Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, 3Deptartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 4Chan Zuckerberg Biohub Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software. Neuroscience Measuring the Influence of Magnetic Vestibular Stimulation on Nystagmus, Self-Motion Perception, and Cognitive Performance in a 7T MRT Gerda Wyssen1,6, Miranda Morrison2, Athanasia Korda2, Wilhelm Wimmer2,3, Jorge Otero-Millan4, Matthias Ertl1, Andreas A. Szukics1,6, Thomas Wyss2, Franca Wagner5,6, Marco D. Caversaccio2,3, Georgios Mantokoudis2,6, Fred W. Mast1,6 1Department of Psychology, University of Bern, 2Department of Otorhinolaryngology, Head and Neck Surgery, lnselspital, University Hospital Bern and University of Bern, 3Hearing Research Laboratory, ARTORG Center for Biomedical Engineering Research, University of Bern, 4Herbert Wertheim School of Optometry and Vision Science, University of California, 5University Department of Diagnostic and Interventional Neuroradiology, Inselspital, Bern University Hospital, University of Bern, 6Translational Imaging Center (TIC), Swiss Institute for Translational and Entrepreneurial Medicine In this article, we describe the experimental setup, material, and procedures to assess reflexive eye movements, self-motion perception, and cognitive tasks under magnetic vestibular stimulation, as well as the anatomical orientation of the vestibular organs, in a 7 Tesla Magnetic resonance tomography (7T-MRT) scanner. Biology Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity Michael Worcester*1, Femila Manoj*1,2, Thomas E. Kuhlman1,2,3 1Department of Physics and Astronomy, University of California, 2Microbiology Program, University of California, 3Biophysics Program, University of California A protocol is outlined to perform live real-time imaging to quantify how the accessory protein TnpB affects the dynamics of transposition in individual live Escherichia coli cells. Biology Forming, Confining, and Observing Microtubule-Based Active Nematics Fereshteh L. Memarian1, Dimitrius A. Khaladj1, Derek Hammar1, Linda S. Hirst1 1Department of Physics, University of California Presented here are methods for preparing active nematics from microtubules and kinesin motors, including protein preparation and construction and the use of wells for active nematic confinement. Cancer Research Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens Jonathan Weitz1, Kevin Christian Montecillo Gulay1, Tatiana Hurtado de Mendoza1, Herve Tiriac1, Joel Baumgartner1, Kaitlyn Kelly1, Jula Veerapong1, Andrew M. Lowy1 1Department of Surgery, University of California We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry. Bioengineering Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements Andre Lai*1, Rachel Rex*2, Kristen L. Cotner1, Alan Dong3, Michael Lustig1,3, Lydia L. Sohn1,2 1Graduate Program in Bioengineering, University of California, Berkeley and University of California, San Francisco, 2Department of Mechanical Engineering, University of California, Berkeley, 3Department of Electrical Engineering and Computer Sciences, University of California, Berkeley Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells. Developmental Biology Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos Sarah Avila-Barnard1, David C. Volz1 1Department of Environmental Sciences, University of California, Riverside This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos. Biochemistry Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction Joann Phan1, Joseph Kapcia III1, Cynthia I. Rodriguez2, Victoria L. Vogel3, Daniel B Cardin3, Sage J. B. Dunham3, Katrine Whiteson1 1Department of Molecular Biology and Biochemistry, University of California Irvine, 2Department of Ecology and Evolutionary Biology, University of California Irvine, 3Entech Instruments Inc. This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing. Behavior Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae Caroline Ackley1, Lindsey Washiashi1, Ruchira Krishnamurthy1, Joel H. Rothman1 1Molecular Cellular and Developmental Biology, University of California The present protocol outlines methods for conducting a large-scale gravitaxis assay with Caenorhabditis dauer larvae. This protocol allows for better detection of gravitaxis behavior compared with a plate-based assay. Immunology and Infection Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria Sydney R. Morrill1,2,3, Kavita Agarwal2,3, Sudeshna Saha2,3, Warren G. Lewis2,3, Nicole M. Gilbert4, Amanda L. Lewis2,3 1Division of Biology and Biomedical Sciences, Washington University School of Medicine, 2Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, 3Glycobiology Research and Training Center, UCSD, 4Department of Pediatrics, Washington University School of Medicine The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis, while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria. Biochemistry Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores Sohee Choi1, Jessica A. San Juan2, Marie C. Heffern2, Michael J. Stevenson1 1Department of Chemistry, University of San Francisco, 2Department of Chemistry, University of California, Davis This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins. Immunology and Infection Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development Sean F. Gilmore*1, Wei He*1, Angela C. Evans1, Delia F. Tifrea2, Sukumar Pal2, Brent Segelke1, Sandra K. G. Peters1, B. Dillon Vannest1, Nicholas O. Fischer1, Amy Rasley1, Luis M. de la Maza2, Matthew A. Coleman1,3 1Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, 2Department of Pathology and Laboratory Medicine, University of California, 3School of Medicine, Radiation Oncology, University of California Davis This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines. Biochemistry Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy Hoang P. M. Nguyen*1, Kelly L. McGuire*1, Brian D. Cook*1, Mark A. Herzik, Jr.1 1Department of Chemistry and Biochemistry, University of California, San Diego This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy. Bioengineering All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System Qin Luo*1, Miao Huang*2, Chenyu Liang2, Justin Zhang3, Gaoming Lin1, Sydney Yu1, Mai Tanaka4,5, Sharon Lepler4,5, Juan Guan5,6,7, Dietmar Siemann4,5, Xin Tang2,5 1Department of Electrical and Computer Engineering, Herbert Wertheim College of Engineering, University of Florida, 2Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering, University of Florida, 3Department of Electrical and Computer Engineering, University of California, 4Department of Radiation Oncology, College of Medicine, University of Florida, 5UF Health Cancer Center, University of Florida, 6Department of Physics, College of Liberal Arts and Sciences, University of Florida, 7Department of Anatomy and Cell Biology, College of Medicine, University of Florida This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP). Developmental Biology Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation Kelly C. Radecki1, Mary Y. Lorenson1, David G. Carter2, Ameae M. Walker1 1Division of Biomedical Sciences, School of Medicine, University of California, Riverside, 2Microscopy and Imaging Core Facility, University of California, Riverside A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells. Biology Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate Robert M. Cooper*1, Taishi Tonooka*1,2, Andriy Didovyk*1,3, Jeff Hasty1,4,5 1Biocircuits Institute, University of California, San Diego, 2Present address, Kyoto Institute of Technology, 3Present address, Vertex Pharmaceuticals, 4Department of Bioengineering, University of California, San Diego, 5Molecular Biology Section, University of California, San Diego This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment. Immunology and Infection Generating Transgenics and Knockouts in Strongyloides Species by Microinjection Michelle L. Castelletto1, Elissa A. Hallem1,2 1Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 2Molecular Biology Institute, University of California, Los Angeles The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S. stercoralis and S. ratti. Developmental Biology Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System Jesselynn LaBelle1, Stephanie Woo1 1Department of Molecular Cell Biology, University of California Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system. 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. Neuroscience A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy Antara Das1, Martin A. Smith2, Diane K. O'Dowd1 1Department of Developmental and Cell Biology, University of California, 2Department of Anatomy and Neurobiology, University of California The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures. Chemistry Gold Nanoparticle Synthesis Jonathan Marrs1, Taher Ghomian2, Lucas Domulevicz1, Cliff McCold3, Joshua Hihath1 1Department of Electrical and Computer Engineering, University of California, Davis, 2Department of Computer Sciences and Electrical Engineering, Marshall University, 3Department of Materials Science and Engineering, University of California, Davis A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene. Genetics Microinjection Method for Anopheles gambiae Embryos Rebeca Carballar-Lejarazú1, Taylor Tushar1, Thai Binh Pham2, Anthony A. James1,2 1Department of Microbiology & Molecular Genetics, University of California, Irvine, 2Department of Molecular Biology & Biochemistry, University of California, Irvine Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes. Genetics Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations Rebeca Carballar-Lejarazú1, Thai Binh Pham2, Adam Kelsey1, Taylor Tushar1, Anthony A. James1,2 1Department of Microbiology & Molecular Genetics, University of California, Irvine, 2Department of Molecular Biology & Biochemistry, University of California, Irvine This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification. Genetics Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes Rebeca Carballar-Lejarazú1, Thai Binh Pham2, Vanessa Bottino-Rojas1, Adriana Adolfi1,3, Anthony A. James1,2 1Department of Microbiology & Molecular Genetics, University of California, Irvine, 2Department of Molecular Biology & Biochemistry, University of California, Irvine, 3Vector Biology Department, Liverpool School of Tropical Medicine The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured. Biology Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning Nathan Grainger1,2, Kenton M. Sanders1, Bernard T. Drumm1,3 1Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, 2Department of Physiology & Membrane Biology, University of California School of Medicine, 3Department of Life & Health Sciences, Dundalk Institute of Technology The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices. Biochemistry Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers Mowei Zhou1, Shadan H. Abdali1, David Dilworth2, Lifeng Liu2, Benjamin Cole2, Neha Malhan1, Amir H. Ahkami1, Tanya E. Winkler1, Joy Hollingsworth3, Julie Sievert3, Jeff Dahlberg3, Robert Hutmacher4,5, Mary Madera6, Judith A. Owiti6, Kim K. Hixson1, Peggy G. Lemaux6, Christer Jansson1, Ljiljana Paša-Tolić1 1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 2DOE-Joint Genome Institute, Lawrence Berkeley Laboratory, 3Kearney Agricultural Research and Extension Center, University of California Agriculture and Natural Resources, 4West Side Research and Extension Center, University of California, 5Department of Plant Sciences, University of California, Davis, 6Department of Plant and Microbial Biology, University of California, Berkeley The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops. Developmental Biology Creating Avian Forebrain Chimeras to Assess Facial Development Diane Hu1, Ralph S. Marcucio1 1Department of Orthopaedic Surgery, Orthopaedic Trauma Institute, University of California This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain during craniofacial development. Bioengineering Generation of Self-assembled Vascularized Human Skin Equivalents Martina M. Sanchez1, Joshua T. Morgan1 1Departments of Bioengineering, University of California The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs. Immunology and Infection Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx Ruichen Sun1, Daniel Brogan1, Anna Buchman1, Ting Yang1, Omar S. Akbari1 1Division of Biological Sciences, Section of Cell and Developmental Biology, University of California This article outlines a detailed protocol for using the RNA-targeting Cas13D enzyme (RfxCas13D) in flies. Genetics Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria Adriana Adolfi1,2, Amy Lynd1, Gareth J. Lycett1, Anthony A. James2,3 1Vector Biology Department, Liverpool School of Tropical Medicine, 2Department of Microbiology & Molecular Genetics, University of California, 3Department of Molecular Biology & Biochemistry, University of California The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the φC31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines. Developmental Biology A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development Renat N. Khaliullin1,2,3, Jeffrey M. Hendel1,2, Adina Gerson-Gurwitz1,2, Shaohe Wang1,4,5, Stacy D. Ochoa1,6, Zhiling Zhao1,7, Arshad Desai1,2, Karen Oegema1,2, Rebecca A. Green1,2 1Ludwig Institute for Cancer Research, San Diego, 2Department of Cellular and Molecular Medicine, University of California, San Diego, 3Recursion Pharmaceuticals, 4Biomedical Sciences Graduate Program, University of California, San Diego, 5Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, 6Department of Biology, San Diego State University, 7Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis. Medicine Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice Shuxun Ren1, Sunny Chang2, Alex Tran3, Arianna Mandelli4, Yibin Wang1, Jessica J. Wang2 1Department of Anesthesiology, University of California, 2Department of Medicine, University of California, 3Department of Microbiology, Immunology & Molecular Genetics, University of California, 4Department of Molecular, Cell, and Developmental Biology, University of California The chronic administration of isoproterenol via an implanted osmotic pump has been used widely to mimic advanced heart failure in mice. Here, we describe detailed methods in surgical mini-pump implantation for the continuous isoproterenol administration over 3 weeks, as well as, echocardiographic assessment for the successful model creation. Bioengineering Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles Gregory Michael Newkirk*1,2, Honghong Wu*1, Israel Santana1, Juan Pablo Giraldo1,2 1Department of Botany and Plant Sciences, University of California, 2Department of Microbiology and Plant Pathology, University of California Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy. Bioengineering A Rapid Image-based Bacterial Virulence Assay Using Amoeba Kumar Perinbam1, Albert Siryaporn1,2 1Department of Physics and Astronomy, University of California, 2Department of Molecular Biology and Biochemistry, University of California Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa. Genetics Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms Behnom Farboud*1,2, Erin Jarvis*1, Theodore L. Roth*3,4,5,6, Jiyung Shin*1,3, Jacob E. Corn1,3, Alexander Marson3,5,6,7,8,9, Barbara J. Meyer1,2, Nipam H. Patel1,10, Megan L. Hochstrasser3 1Department of Molecular Cell Biology, University of California, Berkeley, 2Howard Hughes Medical Institute, University of California, Berkeley, 3Innovative Genomics Institute, University of California, Berkeley, 4Biomedical Sciences Graduate Program, University of California, San Francisco, 5Department of Microbiology and Immunology, University of California, San Francisco, 6Diabetes Center, University of California, San Francisco, 7Chan Zuckerberg Biohub, 8Department of Medicine, University of California, San Francisco, 9UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 10Department of Integrative Biology, University of California, Berkeley Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms. Genetics Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus Ira L. Blitz1 1Department of Developmental and Cell Biology, University of California, Irvine Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated. Genetics Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation Ming Li1,2, Michelle Bui1,2, Omar S. Akbari1,2 1Department of Entomology and Riverside Center of Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, 2Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism. Neuroscience Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease Theresa M. Harrison1, Donald G. McLaren2, Teena D. Moody1, Jamie D. Feusner1, Susan Y. Bookheimer1 1Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, 2Biospective, Inc. This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects. Immunology and Infection Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein Shubhendu Sen Roy1, Huy Cong Xuan Nguyen1, Thomas A. Angelovich2,3, Anna C. Hearps2, Diana Huynh1,4, Anthony Jaworowski2,4, Theodoros Kelesidis1 1University of California, Los Angeles, 2Centre for Biomedical Research, Burnet Institute, 3School of Health and Biomedical Sciences, RMIT University, 4Department of Infectious Diseases, Monash University We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease. Developmental Biology Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue Sumitra Tatapudy1, Marimar Benitez1, Todd Nystul1 1Departments of Anatomy and OB/GYN-RS, University of California, San Francisco We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values. Immunology and Infection Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4 Sharon A. Sagan*1,2, Andrés Cruz-Herranz*1, Collin M. Spencer1,2, Peggy P. Ho3, Lawrence Steinman3, Ari J. Green1, Raymond A. Sobel4, Scott S. Zamvil1,2 1Department of Neurology, University of California, 2Program in Immunology, University of California, 3Department of Neurology and Neurological Sciences, Stanford University, 4Department of Pathology, Stanford University Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction. Biochemistry A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies Prateek Tripathi1, José L. Pruneda-Paz2, Steve A. Kay1 1Department of Neurology, University of Southern California, 2Section of Cell and Developmental Biology, University of California San Diego The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study. Behavior Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm Angela F. Lukowski1, Helen M. Milojevich1 1Department of Psychology and Social Behavior, University of California-Irvine The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood. Neuroscience Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons Ji-Yong Kang1, Daichi Kawaguchi2, Lei Wang3 1Department of Neuroscience, School of Medicine, Tufts University, 2Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 3Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented. Medicine Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment Renske J.E. van den Bijgaart*1, Niwen Kong*1, Carrie Maynard1,2, Vicki Plaks1,3 1Department of Anatomy, University of California, 2Hubrecht Institute-Royal Dutch Academy of Science and University Medical Center Utrecht, 3Department of Cell and Developmental Biology, Sackler Faculty of Medicine We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions. Medicine Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy Yan Wang1,3,4, Dale P. Brown1, Brant D. Watson2, Jeffrey L. Goldberg1,3 1Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 2Departments of Neurology and Biomedical Engineering, University of Miami Miller School of Medicine, 3Shiley Eye Center, University of California, 4Department of Ophthalmology and Vision Science, Fudan University The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases. Developmental Biology Mechanical Vessel Injury in Zebrafish Embryos Hilary Clay1, Shaun R. Coughlin1 1Cardiovascular Research Institute, University of California This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy. Immunology and Infection Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy Caroline Bonnans*1,2, Marja Lohela*2, Zena Werb2 1Department of Oncology, INSERM U661, Functional Genomic Institute, 2Department of Anatomy, University of California By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model. Medicine Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development Daniel P. Vang1, Gregory T. Wurz1, Stephen M. Griffey2, Chiao-Jung Kao1, Audrey M. Gutierrez1, Gregory K. Hanson1, Michael Wolf3, Michael W. DeGregorio1 1Department of Internal Medicine, Division of Hematology and Oncology, University of California, Davis, 2Comparative Pathology Laboratory, UC Davis School of Veterinary Medicine, University of California, Davis, 3Merck Serono Research, Merck KGaA, Darmstadt, Germany An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity. Biology Quantifying Agonist Activity at G Protein-coupled Receptors Frederick J. Ehlert1, Hinako Suga2, Michael T. Griffin3 1Department of Pharmacology, University of California, Irvine, 2Department of Pharmacology, University of California, 3Schmid College of Science, Chapman University A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation. 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 Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA Srikumar Sengupta1, Jennifer M. Bolin1, Victor Ruotti1, Bao Kim Nguyen1, James A. Thomson1,2,3, Angela L. Elwell1, Ron Stewart1 1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts. Bioengineering Adaptation of a Haptic Robot in a 3T fMRI Joseph Snider1, Markus Plank1, Larry May2, Thomas T. Liu2, Howard Poizner3 1Institute for Neural Computation, University of California, 2Department of Radiology, University of California, 3Department of Cognitive Science and Program in Neurosciences, University of California The adaptation and use of a haptic robot in a 3T fMRI is described. Neuroscience Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence Sava Sakadžić1, Emmanuel Roussakis2, Mohammad A. Yaseen1, Emiri T. Mandeville3, Vivek J. Srinivasan1, Ken Arai3, Svetlana Ruvinskaya1, Weicheng Wu1, Anna Devor1,4, Eng H. Lo3, Sergei A. Vinogradov2, David A. Boas1 1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice. Immunology and Infection Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method Hal X. Nguyen1,2,3,4, Kevin D. Beck5, Aileen J. Anderson1,2,3,4,6 1Institute for Memory Impairments and Neurological Disorders, University of California, 2Physical Medicine & Rehabilitation, University of California, 3Anatomy & Neurobiology, University of California, 4Sue and Bill Gross Stem Cell Research Center, University of California, 5Section of Molecular Biology, University of California, 6Reeve-Irvine Research Center, University of California Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord. Medicine A Mouse Model of in Utero Transplantation Amar Nijagal1,2, Tom Le1,2, Marta Wegorzewska1,2,3, Tippi C. MacKenzie1,2,3 1Department of Surgery, University of California, 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, 3Biomedical Sciences Program, University of California The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique Biology Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth Sabrina Lin1,2,3, Shawn Fonteno1, Shruthi Satish1,3, Bir Bhanu4, Prue Talbot1,2 1UCR Stem Cell Center, University of California, 2Department of Cell Biology and Neuroscience, University of California, 3Cell, Molecular, and Developmental Biology Graduate Program, University of California, 4Center for Research in Intelligent Systems, University of California Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.
