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Howard Hughes Medical Institute

31 ARTICLES PUBLISHED IN JoVE

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Biology

Interview: Protein Folding and Studies of Neurodegenerative Diseases
Susan Lindquist 1
1Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

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Biology

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis
Randal Halfmann 1,2,3, Susan Lindquist 1,2,3
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.

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Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila
Claudio Vinegoni 1, Daniel Razansky 2, Chrysoula Pitsouli 3, Norbert Perrimon 3, Vasilis Ntziachristos 2, Ralph Weissleder 1
1Center for Systems Biology, Massachusetts General Hospital, 2Institute for Biological and Medical Imaging (IBMI), Technical University of Munich and Helmholtz Center Munich, 3Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

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Biology

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.
Nynke L. van Berkum *1, Erez Lieberman-Aiden *2,3,4,5, Louise Williams *2, Maxim Imakaev 6, Andreas Gnirke 2, Leonid A. Mirny 3,6, Job Dekker 1, Eric S. Lander 2,7,8
1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University , 5Department of Applied Mathematics, Harvard University , 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

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Biology

Primary Cell Cultures from Drosophila Gastrula Embryos
Norbert Perrimon 1,2, Jonathan Zirin 1, Jianwu Bai 1
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.

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Biology

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice
Yu Chen 1,2, Tambudzai Shamu 2, Hui Chen 3, Peter Besmer 3, Charles L. Sawyers 2,4, Ping Chi 1,5
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.

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Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
Chun-Chun Chen 1, Kazuhiro Wada 2, Erich D. Jarvis 1
1Howard Hughes Medical Institute, Department of Neurobiology, Duke University , 2Department of Biological Sciences, Hokkaido University

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

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Biology

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation
Otto C. Guedelhoefer IV 1,2, Alejandro Sánchez Alvarado 3,4
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.

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Bioengineering

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip
Guido Grossmann 1, Matthias Meier 2,3,4, Heather N. Cartwright 1, Davide Sosso 1, Stephen R. Quake 2,3, David W. Ehrhardt 1, Wolf B. Frommer 1
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.

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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
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.

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Neuroscience

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling
Gustavo Arriaga 1, Joshua J. Macopson 1, Erich D. Jarvis 1,2
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.

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Bioengineering

Sample Drift Correction Following 4D Confocal Time-lapse Imaging
Adam Parslow 1, Albert Cardona 2, Robert J. Bryson-Richardson 1
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.

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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. Fenton 1, Arthur L. Horwich 1,2, Maria Nagy 1,2
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.

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JoVE Journal

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
1Harvard Medical School, 2Division of Hematology/Oncology, Boston Children's Hospital, 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, 4Howard Hughes Medical Institute

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.

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Behavior

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs
Jonathan Chabout 1,2, Joshua Jones-Macopson 1, Erich D. Jarvis 1,2,3
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.

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Developmental Biology

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae
Elliott J. Hagedorn 1,2, Jennifer L. Cillis 3, Caitlyn R. Curley 3, Taylor C. Patch 3, 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
1Division of Hematology/Oncology, Boston Children’s Hospital, 2Harvard Medical School, 3Division 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.

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Genetics

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform
Nicolaas H. Fourie 1, Ralph M. Peace 1,2, Sarah K. Abey 1, LeeAnne B. Sherwin 1, John W. Wiley 3, Wendy A. Henderson 1
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.

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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
1Department of Cardiology, Boston Children's Hospital, 2Department of Pediatrics, Harvard Medical School, 3Department of Genetics, Harvard Medical School, 4Howard Hughes Medical Institute

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

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Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing
Kristin A. Knouse 1,2,3, Jie Wu 4, Austin Hendricks 5
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.

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Neuroscience

Drosophila Courtship Conditioning As a Measure of Learning and Memory
Tom S. Koemans 1,2,3, Cornelia Oppitz 4, Rogier A. T. Donders 5, Hans van Bokhoven 1,3, Annette Schenck 1,3, Krystyna Keleman 6, Jamie M. Kramer 7,8
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.

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Behavior

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila
Sara Batelli 1, Malte Kremer 1,2, Christophe Jung 1, Ulrike Gaul 1
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.

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Neuroscience

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine
Matthew C. Arvin 1, David L. Wokosin 2, Sambashiva Banala 3, Luke D. Lavis 3, Ryan M. Drenan 1
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.

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Biology

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry
Shriram Venkatesan *1, Tejbir S. Kandola *1, Alejandro Rodríguez-Gama 1, Andrew Box 1, Randal Halfmann 1,2
1Stowers Institute for Medical Research, 2Department of Molecular and Integrative Physiology, The University of Kansas School of Medicine

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

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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
1Department of Ophthalmology, Boston Children's Hospital, 2Department of Ophthalmology, Harvard Medical School, 3F.M. Kirby Neurobiology Center, Boston Children's Hospital, 4Department of Neurology, Boston Children's Hospital, 5Department of Neurology, Harvard Medical School, 6Howard Hughes Medical Institute

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.

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Neuroscience

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays
Jason E Chung *1,2, Hannah R Joo *1,2, Clay N Smyth 2, Jiang Lan Fan 3, Charlotte Geaghan-Breiner 2, Hexin Liang 2, Daniel Fan Liu 3, Demetris Roumis 2, Supin Chen 4,5, Kye Y Lee 4, Jeanine A Pebbles 4, Angela C Tooker 4, Vanessa M Tolosa 4,5, Loren M Frank 2,6
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.

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Cancer Research

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses
Kyrie J. Pappas 1, Danielle Choi 1, Charles L. Sawyers 1,2, Wouter R. Karthaus 1
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.

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Neuroscience

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn: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
1Department of Neurology, Boston Children's Hospital, 2FM Kirby Neurobiology Center, Boston Children's Hospital, 3Department of Neurology, Harvard Medical School, 4Medical Genetics Training Program, Harvard Medical School, 5Department of Ophthalmology, Boston Children's Hospital, 6Department of Ophthalmology, Harvard Medical School, 7Broad Institute of M.I.T. and Harvard, 8Howard Hughes Medical Institute, 9Department of Neurology, Kokura Memorial Hospital, 10Department of Genetics, Albert Einstein College of Medicine

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.

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Genetics

R-Loop Analysis by Dot-Blot
Prisila Ramirez 1, Robert J. Crouch 2, Vivian G. Cheung 1,3,4, Christopher Grunseich 5
1Life Sciences Institute, University of Michigan, Ann Arbor, 2Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 3Department of Pediatrics, University of Michigan, Ann Arbor, 4Howard Hughes Medical Institute, 5Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health

This protocol details a simple method that quantifies R-loop, a three-stranded nucleic acid structure that comprises of an RNA-DNA hybrid and a displaced DNA strand.

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Biology

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining
Fengli Guo 1, Melainia McClain 1, Xia Zhao 1, Kexi Yi 1, Tari Parmely 1, Jay Unruh 1, Brian Slaughter 1, Leonid Kruglyak 2,3, Longhua Guo 2,3, Alejandro Sánchez Alvarado 1,2
1Stowers Institute for Medical Research, 2Howard Hughes Medical Institute, 3Department of Human Genetics, University of California Los Angeles

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously inaccessible.

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Genetics

Capturing Chromosome Conformation Across Length Scales
Liyan Yang 1, Betul Akgol Oksuz 1, Job Dekker 1,2, Johan Harmen Gibcus 1
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.

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Bioengineering

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics
Audrey E. Van Heest 1, Feiyang Deng 1, Renee T. Zhao 2, 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
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, 8Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School

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.

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