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The Francis Crick Institute

15 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Studying the Integration of Adult-born Neurons
Yan Gu 1, Stephen Janoschka 1, Shaoyu Ge 1
1Department of Neurobiology & Behavior, State University of New York at Stony Brook

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

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Neuroscience

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation
Emilie Pacary 1, Matilda A. Haas 1, Hendrik Wildner 1, Roberta Azzarelli 1, Donald M. Bell 2, Djoher Nora Abrous 3, François Guillemot 1
1Division of Molecular Neurobiology, MRC National Institute for Medical Research, 2Confocal and Image Analysis Laboratory, National Institute for Medical Research, 3Physiopathologie de la plasticité neuronale, Neurocentre Magendie, Université de Bordeaux

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

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Biology

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
Erik Serrao 1, Peter Cherepanov 2, Alan N. Engelman 1
1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 2Chromatin Structure and Mobile DNA, The Francis Crick Institute

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

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Neuroscience

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ
Tobias Ackels 1,2, Daniela R. Drose 1, Marc Spehr 1
1Department of Chemosensation, Institute for Biology II, RWTH Aachen University, 2Mill Hill Laboratory, The Francis Crick Institute

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

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Immunology and Infection

Optimization of a Quantitative Micro-neutralization Assay
Yipu Lin 1, Yan Gu 1, John W. McCauley 1
1Mill Hill Laboratory, The Francis Crick Institute

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

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Biology

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems
Jean-Philippe Mocho 1, Darren J. Martin 1, Mollie E. Millington 1, Yolanda Saavedra Torres 1
1Biological Research Facility, The Francis Crick Institute

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

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Developmental Biology

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging
Diana Passaro *1, Ander Abarrategi *1, Katie Foster 1, Linda Ariza-McNaughton 1, Dominique Bonnet 1
1Haematopoietic Stem Cell Laboratory, The Francis Crick Institute

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

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Immunology and Infection

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment
Rebeca Jimeno 1,2, Phillip M. Brailey 1,2, Patricia Barral 1,2
1The Peter Gorer Department of Immunobiology, King's College London, 2The Francis Crick Institute

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

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Neuroscience

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains
Myrto Denaxa 1,2, Guilherme Neves 3, Juan Burrone 3, Vassilis Pachnis 1
1Nervous System Development and Homeostasis Laboratory, The Francis Crick Institute, 2Neuroscience Centre, Biomedical Sciences Research Centre "Al. Fleming", 3Centre for Developmental Neurobiology, King's College London

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

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Developmental Biology

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages
Federica Mangione 1,2, Enrique Martin-Blanco 1
1Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Parc Científic de Barcelona, 2The Francis Crick Institute

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

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Immunology and Infection

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells
Paula Ordonez 1, Kate N. Bishop 2, Jonathan P. Stoye 1, Harriet Cordelia Theed Groom 3
1Retrovirus-Host Interactions Laboratory, The Francis Crick Institute, 2Retroviral Replication Laboratory, The Francis Crick Institute, 3Sidney Sussex College, Department of Medicine, University of Cambridge

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

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Neuroscience

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche
Thomas Kerloch 1, Tjaša Lepko 2, Kirill Shkura 2, François Guillemot 1, Sébastien Gillotin 2
1The Francis Crick Institute, 2MSD R&D Innovation Centre

Presented here is a method to sequence single nuclei isolated from the mouse dentate gyrus that excludes most neurons through fluorescence-activated nuclei (FAN)-sorting. This approach generates high-quality expression profiles and facilitates the study of most other cell types represented in the niche, including scarce populations such as neural stem cells.

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Biology

Imaging ATG9A, a Multi-Spanning Membrane Protein
Alexander R. van Vliet *1,2, Stefano De Tito *1, Eugenia Almacellas *1, Sharon A. Tooze 1
1Molecular Cell Biology of Autophagy, The Francis Crick Institute, 2MRC Laboratory of Molecular Biology

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

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Bioengineering

A Practical Guide to Phage- and Robotics-Assisted Near-Continuous Evolution
Samir Aoudjane 1, Stefan Golas 2, Osaid Ather 1, Michael J. Hammerling 3, Erika DeBenedictis 1
1The Francis Crick Institute, 2Massachusetts Institute of Technology, 3Future House

Phage- and Robotics-assisted Near-continuous Evolution (PRANCE) is a technique for rapid, robust protein evolution. Robotics allows the parallelization of experiments, real-time monitoring, and feedback control.

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Biochemistry

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase
Cameron McClymont 1, Karolina Chabowska 1, Sherry Xie 1, May Thu Kyaw 1, Hasan Yardimci 1
1The Francis Crick Institute

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

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