Howard Hughes Medical Institute View Institution's Website 27 articles published in JoVE Bioengineering CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics Audrey E. Van Heest1, Feiyang Deng1, Renee T. Zhao2, Nour Saida Harzallah2,3, Heather E. Fleming3,4, Sangeeta N. Bhatia2,3,4,5,6,7,8, Liangliang Hao1,2,3 1Department of Biomedical Engineering, Boston University, 2Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Howard Hughes Medical Institute, 5Broad Institute of Massachusetts Institute of Technology and Harvard, 6Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 7Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 8 This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities. Genetics Capturing Chromosome Conformation Across Length Scales Liyan Yang1, Betul Akgol Oksuz1, Job Dekker1,2, Johan Harmen Gibcus1 1Department of Systems Biology, University of Massachusetts Medical School, 2Howard Hughes Medical Institute Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection. Neuroscience Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice Ryosuke Fujiki1,2,3,4,9, Joun Y. Lee1,2,10, Julie A. Jurgens1,2,3,7, Mary C. Whitman2,5,6, Elizabeth C. Engle1,2,3,4,5,6,7,8 1 This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons. Cancer Research Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses Kyrie J. Pappas1, Danielle Choi1, Charles L. Sawyers1,2, Wouter R. Karthaus1 1Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 2Howard Hughes Medical Institute Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Neuroscience Chronic Implantation of Multiple Flexible Polymer Electrode Arrays Jason E Chung*1,2, Hannah R Joo*1,2, Clay N Smyth2, Jiang Lan Fan3, Charlotte Geaghan-Breiner2, Hexin Liang2, Daniel Fan Liu3, Demetris Roumis2, Supin Chen4,5, Kye Y Lee4, Jeanine A Pebbles4, Angela C Tooker4, Vanessa M Tolosa4,5, Loren M Frank2,6 1Medical Scientist Training Program and Neuroscience Graduate Program, University of California San Francisco, 2Kavli Institute for Fundamental Neuroscience, Center for Integrative Neuroscience, and Department of Physiology, University of California San Francisco, 3Bioengineering Graduate Program, University of California San Francisco, 4Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory, 5Neuralink Corp., 6Howard Hughes Medical Institute Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species. Neuroscience Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth Mary C. Whitman1,2,3, Jessica L. Bell1,3, Elaine H. Nguyen1,3, Elizabeth C. Engle1,2,3,4,5,6 1 An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media. Neuroscience Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine Matthew C. Arvin1, David L. Wokosin2, Sambashiva Banala3, Luke D. Lavis3, Ryan M. Drenan1 1Department of Pharmacology, Northwestern University Feinberg School of Medicine, 2Department of Physiology, Northwestern University Feinberg School of Medicine, 3Janelia Research Campus, Howard Hughes Medical Institute This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology. Behavior Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila Sara Batelli1, Malte Kremer1,2, Christophe Jung1, Ulrike Gaul1 1Gene Center and Department of Biochemistry, Ludwig-Maximilians-University Munich, 2Janelia Farm Research Campus, Howard Hughes Medical Institute Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system. Developmental Biology Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos Lorena Benedetti1,2,3, Anna Ghilardi4, Laura Prosperi4, Maura Francolini*1, Luca Del Giacco*4 1Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 2Department of Neuroscience; Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, 3Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, 4Department of BioSciences, Università degli Studi di Milano Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest. Neuroscience Drosophila Courtship Conditioning As a Measure of Learning and Memory Tom S. Koemans1,2,3, Cornelia Oppitz4, Rogier A. T. Donders5, Hans van Bokhoven1,3, Annette Schenck1,3, Krystyna Keleman6, Jamie M. Kramer7,8 1Department of Human Genetics, Radboud University Medical Center, 2Radboud Institute of Molecular Life Sciences, Radboud University, 3Donders Institute for Brain, Cognition, and Behaviour, Centre for Neuroscience, Radboud University, 4Research Institute of Molecular Pathology, Vienna, Austria, 5Department for Health Evidence, Radboud University Medical Center, 6Janelia Research Campus, Howard Hughes Medical Institute, 7Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, 8Department of Biology, Faculty of Science, Western University This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory. Behavior Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs Jonathan Chabout1,2, Joshua Jones-Macopson1, Erich D. Jarvis1,2,3 1Department of Neurobiology, Duke University, 2Howard Hughes Medical Institute, 3The Rockefeller University Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs. Genetics Detection of Copy Number Alterations Using Single Cell Sequencing Kristin A. Knouse1,2,3, Jie Wu4, Austin Hendricks5 1Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, 2Howard Hughes Medical Institute, 3Division of Health Sciences and Technology, Harvard Medical School, 4The Barbara K. Ostrom (1978) Bioinformatics and Computing Facility in the Swanson Biotechnology Center, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 5BioMicro Center, Department of Biology, Massachusetts Institute of Technology Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells. Genetics Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts Jian Ding1,2, Zhi-Qiang Lin1,2, Jian-Ming Jiang3,4, Christine E. Seidman3,4, Jonathan G. Seidman3,4, William T. Pu1,2, Da-Zhi Wang1,2 1 In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described. Genetics Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform Nicolaas H. Fourie1, Ralph M. Peace1,2, Sarah K. Abey1, LeeAnne B. Sherwin1, John W. Wiley3, Wendy A. Henderson1 1Digestive Disorders Unit, National Institute of Nursing Research, National Institutes of Health, DHHS, 2National Institutes of Health Research Scholar, Howard Hughes Medical Institute, 3Internal Medicine, Medical School, University of Michigan We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome. Developmental Biology Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae Elliott J. Hagedorn1,2, Jennifer L. Cillis3, Caitlyn R. Curley3, Taylor C. Patch3, Brian Li1,2, Bradley W. Blaser1,2,7, Raquel Riquelme1,2, Leonard I. Zon1,2,4,5,6, Dhvanit I. Shah1,2,3,4,5 1Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest. Neuroscience Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling Gustavo Arriaga1, Joshua J. Macopson1, Erich D. Jarvis1,2 1Department of Neurobiology, Duke University Medical Center, 2Howard Hughes Medical Institute Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons. Biology Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 Daniel E. Bauer*1,2,3, Matthew C. Canver*1, Stuart H. Orkin1,2,3,4 1Harvard Medical School, 2 CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9. Bioengineering Sample Drift Correction Following 4D Confocal Time-lapse Imaging Adam Parslow1, Albert Cardona2, Robert J. Bryson-Richardson1 1School of Biological Sciences, Monash University, 2Janelia Farm Research Campus, Howard Hughes Medical Institute Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time. Neuroscience Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection Urmi Bandyopadhyay1,2, Wayne A. Fenton1, Arthur L. Horwich1,2, Maria Nagy1,2 1Department of Genetics, Yale School of Medicine, 2Howard Hughes Medical Institute High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol. Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR. Immunology and Infection Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria Tal Danino*1, Arthur Prindle*2, Jeff Hasty2,3,4, Sangeeta Bhatia1,5,6,7,8 1Health Sciences and Technology, Massachusetts Institute of Technology, 2Department of Bioengineering, University of California, San Diego, 3Biocircuits Institute, University of California, San Diego, 4Molecular Biology Section, Division of Biological Science, University of California, San Diego, 5Broad Institute of Harvard and MIT, 6Department of Medicine, Brigham and Women's Hospital, 7Electrical Engineering and Computer Science and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 8Howard Hughes Medical Institute The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting. Biology Planarian Immobilization, Partial Irradiation, and Tissue Transplantation Otto C. Guedelhoefer IV1,2, Alejandro Sánchez Alvarado3,4 1Department of Neurobiology and Anatomy, University of Utah School of Medicine, 2Department of Molecular, Cellular and Developmental Biology, UCSB, 3Howard Hughes Medical Institute, 4Stowers Institute for Medical Research An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals. Bioengineering Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip Guido Grossmann1, Matthias Meier2,3,4, Heather N. Cartwright1, Davide Sosso1, Stephen R. Quake2,3, David W. Ehrhardt1, Wolf B. Frommer1 1Department of Plant Biology, Carnegie Institution for Science, 2Howard Hughes Medical Institute, 3Departments of Applied Physics and Bioengineering, Stanford University, 4Department of Microsystems Engineering (IMTEK) and Center for Biological Signaling Studies (BIOSS), University of Freiburg This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels. Neuroscience Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents Marie Vandecasteele1,2, S. M.1, Sébastien Royer1,3, Mariano Belluscio1, Antal Berényi1, Kamran Diba1,4, Shigeyoshi Fujisawa1, Andres Grosmark1, Dun Mao1, Kenji Mizuseki1, Jagdish Patel1, Eran Stark1, David Sullivan1, Brendon Watson1, György Buzsáki1 1Center for Molecular and Behavioral Neuroscience, University of New Jersey, 2Center for Interdisciplinary Research in Biology, Collège de France, 3Janelia Farm Research Campus, Howards Hughes Medical Institute, 4Deptartment of Psychology, University of Wisconsin at Milwaukee We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication. Biology Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice Yu Chen1,2, Tambudzai Shamu2, Hui Chen3, Peter Besmer3, Charles L. Sawyers2,4, Ping Chi1,5 1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks. Biology Primary Cell Cultures from Drosophila Gastrula Embryos Norbert Perrimon1,2, Jonathan Zirin1, Jianwu Bai1 1Department of Genetics, Harvard Medical School, 2Howard Hughes Medical Institute We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells. Biology Spinal Cord Electrophysiology Allyn Meyer1, Benjamin W. Gallarda1,2, Samuel Pfaff1, William Alaynick1 1The Salk Institute for Biological Studies, Howard Hughes Medical Institute and Gene Expression Laboratory, 2Biology Graduate Program, University of California San Diego - UCSD A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies. Biology Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis Randal Halfmann1,2,3, Susan Lindquist1,2,3 1Whitehead Institute for Biomedical Research, 2Department of Biology, MIT - Massachusetts Institute of Technology, 3Howard Hughes Medical Institute SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.