Howard Hughes Medical Institute

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 Harzallah^2,3, Heather E. Fleming^3,4, Sangeeta N. Bhatia^{2,3,4,5,6,7,8}, Liangliang Hao^{1,2,3 1}Department 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 Dekker^1,2, Johan Harmen Gibcus^{1 1}Department 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 Isl^mn:GFP Transgenic Mice Ryosuke Fujiki^1,2,3,4,9, Joun Y. Lee^1,2,10, Julie A. Jurgens^1,2,3,7, Mary C. Whitman^2,5,6, Elizabeth C. Engle^{1,2,3,4,5,6,7,8 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. Sawyers^1,2, Wouter R. Karthaus^{1 1}Human 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 Chen^4,5, Kye Y Lee⁴, Jeanine A Pebbles⁴, Angela C Tooker⁴, Vanessa M Tolosa^4,5, Loren M Frank^{2,6 1}Medical 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. Whitman^1,2,3, Jessica L. Bell^1,3, Elaine H. Nguyen^1,3, Elizabeth C. Engle^{1,2,3,4,5,6 1} An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 Isl^MN: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. Drenan^{1 1}Department 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 Kremer^1,2, Christophe Jung¹, Ulrike Gaul^{1 1}Gene 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 Benedetti^1,2,3, Anna Ghilardi⁴, Laura Prosperi⁴, Maura Francolini*¹, Luca Del Giacco*^{4 1}Department 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. Koemans^1,2,3, Cornelia Oppitz⁴, Rogier A. T. Donders⁵, Hans van Bokhoven^1,3, Annette Schenck^1,3, Krystyna Keleman⁶, Jamie M. Kramer^{7,8 1}Department 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 Chabout^1,2, Joshua Jones-Macopson¹, Erich D. Jarvis^{1,2,3 1}Department 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. Knouse^1,2,3, Jie Wu⁴, Austin Hendricks^{5 1}Koch 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 Ding^1,2, Zhi-Qiang Lin^1,2, Jian-Ming Jiang^3,4, Christine E. Seidman^3,4, Jonathan G. Seidman^3,4, William T. Pu^1,2, Da-Zhi Wang^{1,2 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. Peace^1,2, Sarah K. Abey¹, LeeAnne B. Sherwin¹, John W. Wiley³, Wendy A. Henderson^{1 1}Digestive 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. Hagedorn^1,2, Jennifer L. Cillis³, Caitlyn R. Curley³, Taylor C. Patch³, Brian Li^1,2, Bradley W. Blaser^1,2,7, Raquel Riquelme^1,2, Leonard I. Zon^1,2,4,5,6, Dhvanit I. Shah^{1,2,3,4,5 1}Division 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. Jarvis^{1,2 1}Department 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. Orkin^{1,2,3,4 1}Harvard 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-Richardson^{1 1}School 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 Bandyopadhyay^1,2, Wayne A. Fenton¹, Arthur L. Horwich^1,2, Maria Nagy^{1,2 1}Department 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 Hasty^2,3,4, Sangeeta Bhatia^{1,5,6,7,8 1}Health 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 IV^1,2, Alejandro Sánchez Alvarado^{3,4 1}Department 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 Meier^2,3,4, Heather N. Cartwright¹, Davide Sosso¹, Stephen R. Quake^2,3, David W. Ehrhardt¹, Wolf B. Frommer^{1 1}Department 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 Vandecasteele^1,2, S. M.¹, Sébastien Royer^1,3, Mariano Belluscio¹, Antal Berényi¹, Kamran Diba^1,4, Shigeyoshi Fujisawa¹, Andres Grosmark¹, Dun Mao¹, Kenji Mizuseki¹, Jagdish Patel¹, Eran Stark¹, David Sullivan¹, Brendon Watson¹, György Buzsáki^{1 1}Center 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 Chen^1,2, Tambudzai Shamu², Hui Chen³, Peter Besmer³, Charles L. Sawyers^2,4, Ping Chi^{1,5 1}Department 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 Perrimon^1,2, Jonathan Zirin¹, Jianwu Bai^{1 1}Department 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. Gallarda^1,2, Samuel Pfaff¹, William Alaynick^{1 1}The 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 Halfmann^1,2,3, Susan Lindquist^{1,2,3 1}Whitehead 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.