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.
A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.
1Department of Internal Medicine D, Experimental Nephrology, University of Münster, 2Department of Nuclear Medicine, University of Münster, 3European Institute for Molecular Imaging, University of Münster
We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.
1Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, 2UCLA AIDS Institute, 3Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, 4Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, 5Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA
The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.
Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)
1Division of Cardiovascular Medicine, University of Pennsylvania, Perelman School of Medicine, 2Department of Radiology, University of Pennsylvania, Perelman School of Medicine, 3Department of Dermatology, University of Pennsylvania, Perelman School of Medicine
There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.
A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.
Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.
This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle’s contractile response at 37 degrees C with an intact circulation.
1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California at Los Angeles, 2Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California at Los Angeles, 3California NanoSystems Institute, University of California at Los Angeles, 4Nuclear Medicine, PET Center, Shanghai Medical Collegea, Fudan University, 5Electronics and Information Engineering, College of Electronics and Information Engineering, Wuhan Textile University
A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).
The current article describes the basics of multivariate analysis and contrasts it to the more commonly used voxel-wise univariate analysis. Both types of analysis are applied to a clinical-neuroscience data set. Supplementary split-half simulations show better replication of the multivariate results in independent data sets.
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Back in 1905, in what is now the Czech Republic, Eduard Zirm performed the first corneal transplantation surgery (keratoplasty), which restored vision to a patient blinded by corneal injury. Today, eye banks all over the world prepare, store, and distribute donated corneas to hospitals so that thousands of sight-saving keratoplasties can be performed every year. In June 2012, JoVE has its eye on two research groups, one from Italy and the other from Michigan, who demonstrate two distinct methods for corneal graft preparation prior to transplantation.
With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.
1Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 2Lab. PALM, Université de Caen Basse-Normandie, 3Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children's Hospital, Harvard Medical School, 4ISS, INC.
We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.
In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.
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.
Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.
A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.
Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc
Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.
1Centre of Excellence for Alzheimer's Disease Research and Care, School of Medical sciences, Edith Cowan University, 2Centre for Clinical Research in Neuropsychiatry, Graylands Hospital, University of Western Australia, 3McCusker Alzheimer's Research foundation, 4School of Medicine and Pharmacology, University of Western Australia, 5Department of Molecular and Biomedical Sciences, University of Adelaide, 6School of Biomedical Sciences, Curtin University of Technology, 7School of Psychiatry and Clinical Neurosciences, University of Western Australia
This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.
The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli
The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.
1Department of Radiation Oncology, University Hospitals Case Medical Center and Case Western Reserve University School of Medicine, 2Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University Hospitals Case Medical Center and Case Western Reserve University School of Medicine
Stereotactic body radiotherapy (SBRT) involves image-guided, ablative radiation delivered to cancer targets refractory to chemotherapy or to conventional radiation treatment. The robotic-armed Cyberknife SBRT system, using sophisticated target localization, delivers hypofractionated radiation doses capable of sterilizing cancer targets. This article will consider new therapeutic roles of SBRT for gynecological cancers.
Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 μm) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.
Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.
A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.
Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells
The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.
The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.
A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.
This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.
1Department of Medical Biophysics, University of Western Ontario, 2London Regional Cancer Program, London Health Science Centre, 3Department of Pathology, Vanderbilt University, 4Translational Prostate Cancer Research Group, London Health Science Centre
We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.
Basic Surgical Techniques in the Göttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion
1Department of Neurosurgery, Aarhus University Hospital, 2Department of Neurobiology, Institute of Anatomy, Faculty of Health Sciences, Aarhus University, 3Positron Emission Tomography (PET) Centre, Aarhus University Hospital
The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of Göttingen minipigs in biomedical research.
We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.
1Fischell Department of Bioengineering, University of Maryland, 2Institute for Bioscience and Biotechnology Research, University of Maryland, 3Department of Materials Science and Engineering, University of Maryland
This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.
This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.
TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination
A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.
Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature
1U.S. Department of Agriculture, 2Department of Viticulture and Enology, University of California - Davis, 3Hawkesbury Institute for the Environment, University of Western Sydney, 4Advanced Light Source, Lawrence Berkeley National Lab, 5Citrus Research & Education Center, University of Florida
High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.
Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.
An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.
A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.
1Institute for Neural Computation, University of California, 2Department of Radiology, University of California, 3Department of Cognitive Science and Program in Neurosciences, University of California
The adaptation and use of a haptic robot in a 3T fMRI is described.
This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.
Neuroimaging techniques, such as functional MRI and Diffusion Tensor Imaging have become increasingly useful in characterizing the cognitive and neural deficits in autism. An examination of brain connectivity in autism at a network level along with adaptations for scanning children with developmental disabilities is presented.
In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.
MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.
Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology
1Graduate Center for Gerontology, University of Kentucky College of Public Health, 2Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, 3Sanders-Brown Center on Aging, University of Kentucky College of Medicine
This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer’s disease.
A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells
This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.
1Department of Pharmacology, Vanderbilt University, 2Vanderbilt Center for Bone Biology, Vanderbilt University, 3Department of Veterans Affairs, Tennessee Valley Healthcare System (VISN 9), 4Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University, 5Department of Cancer Biology, Vanderbilt University
Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.
Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.
Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling
A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.
Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions
1Institute of Movement and Neurosciences, German Sport University Cologne, 2Deptartment of Surgical Skills, University of Toronto, 3School of Human Movement Studies, Institute of Health and Biomedical Innovation, Queensland University of Technology, 4Brain Products GmbH, Scientific Support, Gilching, Germany
The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.
1Department of Materials Science and Engineering, MIT - Massachusetts Institute of Technology, 2Department of Mechanical Engineering, MIT - Massachusetts Institute of Technology, 3HST Center for Biomedical Engineering and Harvard Stem Cell Institute, Brigham and Women's Hospital and Harvard Medical School
We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.