Epigenomics:
The systematic study of the global gene expression changes due to Epigenetic processes and not due to DNA base sequence changes.
JoVE General
A Rapid Technique for the Visualization of Live Immobilized Yeast Cells
Department of Molecular Biology, Princeton University
A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR
JoVE General
Methylated DNA Immunoprecipitation
1Department of Cancer Genetics and Developmental Biology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3These authors contributed equally., 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC, 5Photography/Video Production, Multi-Media Services, BC Cancer Agency, 6Department of Medical Genetics, Life Sciences Institute,, University of British Columbia - UBC
This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).
JoVE General
Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
1Department of Human Genetics, Emory University School of Medicine, 2Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago
Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.
JoVE Clinical and Translational Medicine
A Zebrafish Model of Diabetes Mellitus and Metabolic Memory
1Dr. William M. Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science, 2Chicago Medical School, Rosalind Franklin University of Medicine and Science
Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.
JoVE Neuroscience
Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
Max Planck Institute of Psychiatry
A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.
JoVE General
A Chromatin Assay for Human Brain Tissue
Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School
Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.
JoVE General
Chromatin Immunoprecipitation (ChIP) using Drosophila tissue
Department of Biology, Johns Hopkins University
Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.
JoVE General
Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology
This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.
JoVE General
Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University
This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.
JoVE Immunology and Infection
Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT
1 , Whitehead Institute for Biomedical Research, 2Departments of Microbiology and Biological Sciences, National University of Singapore, 3Department of Biology, Massachusetts Institute of Technology
We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.
JoVE Neuroscience
Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury
1Laboratory for NeuroRegeneration and Repair, Department of Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 2Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen
We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.
JoVE Editorial
November 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
In this issue, Oestreicher et al. show us how to isolate magnetotactic bacteria from freshwater samples, and concentrate the bacteria at one end of a glass capillary. The magnetotactic bacteria can then be visualized by light and transmission electron microscopy, and used for various other assays.
JoVE General
Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences
We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.
JoVE Clinical and Translational Medicine
Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens
1Department of Neurological Surgery, The Ohio State University Medical Center, 2Department of Pathology, The Ohio State University Medical Center
Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed “tumor explant method”. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.
JoVE General
Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation
1Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, University of Melbourne
Microscopic analysis of γH2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced γH2AX formation within the nucleus.
JoVE General
Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization
1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine
Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).
JoVE General
Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA
1Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 2Department of Molecular and Cell Biology, University of Connecticut, 3Department of Pharmaceutical Sciences, University of Connecticut
Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.
JoVE General
An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
Wellcome Trust Centre for Human Genetics, University of Oxford
Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.
JoVE General
Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats
1Department of Oncology, Georgetown University, 2Institute of Biomedicine, University of Turku Medical Faculty
Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.
JoVE General
Isolation and Derivation of Mouse Embryonic Germinal Cells
The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.
JoVE Clinical and Translational Medicine
Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue
1Strategic Research Center for Stem Cell Biology and Cell Therapy, University of Lund, 2Department of Cardiology Lund University Hospital, University of Lund
Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.
JoVE Immunology and Infection
Isolation of Precursor B-cell Subsets from Umbilical Cord Blood
1Department of Pathology and Anatomical Sciences, University of Missouri-Columbia, 2Laboratory for Infectious Disease Research, University of Missouri-Columbia
Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.
JoVE General
Generation of Mice Derived from Induced Pluripotent Stem Cells
1Dorris Neuroscience Center & Department of Cell Biology, The Scripps Research Institute, 2Mouse Genetics Core Facility, The Scripps Research Institute
Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.
JoVE General
Neuronal Nuclei Isolation from Human Postmortem Brain Tissue
Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School
The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.
JoVE General
Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme
1Department of Craniofacial Biology, University of Colorado Denver Anschutz Medical Campus, 2Department of Cell and Developmental Biology, University of Colorado Denver Anschutz Medical Campus
A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.
JoVE General
DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC
This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.
JoVE Neuroscience
High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue
Applications and Product Development, New England Biolabs
The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing β-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.
JoVE General
DNA Methylation: Bisulphite Modification and Analysis
1Epigenetics Group, Cancer Research Program, Garvan Institute of Medical Research, 2St Vincent's Clinical School, University of NSW
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
JoVE General
Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets
1Department of Biochemistry and Molecular Genetics, University of Illinois Chicago - UIC, 2Research Unit on Biomedical Informatics, Universitat Pompeu Fabra, 3Genome Technology Core, Whitehead Institute for Biomedical Research
Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.
JoVE General
Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer
1Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, 2Southern Arizona Veterans Affairs Health Care System, Tucson, AZ, 3Department of Surgery, College of Medicine, University of Arizona, Tucson, 4Biomedical Diagnostics and Research, Tucson, AZ, 5Department of Medicine, College of Medicine, University of Arizona, Tucson
Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.
JoVE General
Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector
1Center for Regenerative Medicine (CReM), Boston University School of Medicine, 2Department of Hematology, Children's Hospital of Philadelphia, 3Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia
Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.
JoVE Immunology and Infection
Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation
Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School
Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.
JoVE General
Detection of Histone Modifications in Plant Leaves
1Department of Botany, RWTH Aachen University, 2Department of Plant Physiology, RWTH Aachen University, 3Department of Botany, Leibniz University
A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.
JoVE General
Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation
1Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, 2A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, 3Department of Biochemistry, National University of Singapore, Singapore
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.
JoVE General
Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass
Department of Pathology, University of Utah School of Medicine
We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.
JoVE General
Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts
1Department of Molecular Carcinogenesis and Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center, 2Department of Cell Biology, Poznan University of Medical Sciences, 3Department of Molecular Biology, The Scripps Research Institute
We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.
JoVE General
Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP
Yale Stem Cell Center, Department of Genetics, Yale School of Medicine
A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.
JoVE Immunology and Infection
Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy
This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.
JoVE General
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences
We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.
JoVE General
Chromatin Isolation by RNA Purification (ChIRP)
ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.
JoVE General
Single Oocyte Bisulfite Mutagenesis
1Department of Obstretrics & Gynaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Children's Health Research Institute
Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.
JoVE General
Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization
A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.
JoVE General
Nuclear Transfer into Mouse Oocytes
Department of Molecular and Cell Biology, Harvard
This movie and the protocol are intended to help learning nuclear transfer.
JoVE General
Chromatin Immunoprecipitation from Human Embryonic Stem Cells
Department of Biochemistry, University of California - Riverside
The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.
JoVE General
Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm
Department of Biology, Saint Louis University
Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.
JoVE General
Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF
We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.
JoVE Neuroscience
Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF
We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.
JoVE Neuroscience
A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field
1Institute for Biomaterials and Biomedical Engineering, University of Toronto, 2Lyndhurst Centre, Toronto Rehabilitation Institute, 3Department of Surgery, University of Toronto
In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.
JoVE General
Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures
Stem Cell Research Unit, Medical University of Graz, Austria
Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.
JoVE Clinical and Translational Medicine
Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture
Life Science Division, Lawrence Berkeley National Laboratory
A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.
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