1Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center
Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.
Published February 9, 2015. Keywords: Molecular Biology, human EF1α, promoter, SV40 promoter, CMV promoter, Gibson assembly, embryonic stem cells, protein expression, FLAG-tag, synthetic biology
1Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Systems Biology, Harvard Medical School, 3Department of Biotechnology, Delft University of Technology
This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.
Published April 18, 2014. Keywords: Environmental Sciences, Plant Leaves, Synthetic Biology, Plants, Genetically Modified, DNA, Plant, RNA, Gene Targeting, Plant Physiological Processes, Genes, Gene gun, Gibson assembly, Nicotiana benthamiana, Alternative splicing, confocal microscopy, chloroplast, peroxisome
1Institute of Chemistry and The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem
This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.
Published November 21, 2013. Keywords: Chemistry, Materials (General), self-assembly, peptides, diphenylalanine, atomatic interactions, coassembly, molecular recognition
1Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 2Department of Chemistry, The Johns Hopkins University
We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.
Published February 4, 2013. Keywords: Chemistry, Chemical Engineering, Biomolecular Engineering, Materials Science, Physics, Nanotechnology, Molecular Self-assembly, Electrochemistry, Folding, three dimensional, lithography, colloid, patchy particles, particles, nanoparticles, robotics, drug delivery, microfabrication, nanofabrication, nano, assembly, synthesis, reaction, origami
1Department of Biomedical Engineering, Carnegie Mellon University, 2Department of Materials Science and Engineering, Carnegie Mellon University
A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.
Published April 17, 2014. Keywords: Bioengineering, Nanofibers, Nanofabrics, Extracellular Matrix Proteins, Microcontact Printing, Fibronectin, Laminin, Tissue Engineering, poly(N-isopropylacrylamide), Surface-Initiated Assembly
1Department of Chemistry, Gottwald Center for the Sciences, University of Richmond, 2Department of Biochemistry and Molecular Biology, Gottwald Center for the Sciences, University of Richmond
Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).
Published October 4, 2011. Keywords: Bioengineering, Monolayer protected clusters, film assemblies, protein monolayer electrochemistry, azurin, self-assembled monolayers
JoVE Immunology and Infection
1Genetics and Biochemistry Branch of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution “snapshots” of an ordered assembly pathway in a living cell.
Published December 17, 2013. Keywords: Immunology, Autotransporters, Bam complex, Molecular chaperones, protein-protein interactions, Site-specific photocrosslinking
1Department of Electrical and Computer Engineering, University of Minnesota, 2Department of Biomedical Engineering, University of Minnesota, 3Department of Neurology, Mayo Clinic College of Medicine, 4Department of Immunology, Mayo Clinic College of Medicine
Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.
Published May 8, 2014. Keywords: Bioengineering, supported lipid bilayer, beads, microarray, fluorescence, microfabrication, nanofabrication, atomic layer deposition, myelin, lipid rafts
1Biochemistry and Molecular Biology, Colorado State University
A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA. We also describe how sedimentation velocity experiments in the analytical ultracentrifuge, and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.
Published September 10, 2013. Keywords: Cellular Biology, Chromosome Structures, Chromatin, Nucleosomes, Histones, Microscopy, Atomic Force (AFM), Biochemistry, Chromatin, Nucleosome, Nucleosomal Array, Histone, Analytical Ultracentrifugation, Sedimentation Velocity
1Center for Brain Science, Harvard University, 2Program in Neuroscience, Harvard University, 3Department of Organismic and Evolutionary Biology, Harvard University
The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.
Published November 8, 2012. Keywords: Neuroscience, Physiology, Medicine, Anatomy, Mechanical Engineering, microdrive, in-vivo chronic recording, electrophysiology, songbirds
JoVE Immunology and Infection
1Department of Plant Pathology and Microbiology, University of California, Riverside, 2Department of Bioengineering, University of California, Riverside
A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.
Published March 1, 2012. Keywords: Immunology, Agrobacterium, Brome mosaic virus, Nicotiana benthamiana, encapsidation, dissociation, in vitro assembly, Nano technology
1National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science
The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.
Published November 5, 2009. Keywords: Basic Protocols, analytical ultracentrifugation, sedimentation velocity, sedimentation equilibrium, protein characterization, sedimentation coefficient
1Biomedical Engineering Department, Worcester Polytechnic Institute
This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.
Published November 25, 2011. Keywords: Bioengineering, Cell-derived matrix, vascular tissue engineering, smooth muscle cells, cellular self-assembly, tissue biomechanics
1Mechanical Engineering, Johns Hopkins University, 2Biomedical Engineering, Duke University, 3Biomedical Engineering, Johns Hopkins University
We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.
Published August 26, 2009. Keywords: Biomedical Engineering, microfluidics, gene delivery, quantum dots, fluorescence resonance energy transfer, self-assembly, nanocomplexes
1Molecular Foundry, Lawrence Berkeley National Laboratory
A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.
Published November 2, 2011. Keywords: Bioengineering, Biomimetic polymer, peptoid, nanosheet, solid-phase synthesis, self-assembly, bilayer
1Neurosurgery, Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center, 3Molecular & Cellular Biology, Baylor College of Medicine
We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.
Published January 11, 2012. Keywords: Bioengineering, molecular machine, bio-nanotechnology, 5'OH DNA breaks, 5'PO4 DNA breaks, apoptosis labeling, in situ detection, vaccinia topoisomerase I, DNA breaks, green nanotechnology
1Department of Brain and Cognitive Science, MIT - Massachusetts Institute of Technology, 2Picower Institute for Learning and Memory, MIT - Massachusetts Institute of Technology
In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.
Published April 22, 2009. Keywords: Neuroscience, fabrication, micro-drive array, tetrode, electrophysiology, multiple neuronal recordings, in vivo recording, systems neuroscience, hippocampus, coordinated neural activity, cortex, rat brain
1Department of Developmental and Cell Biology, University of California, Irvine (UCI)
This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.
Published April 29, 2007. Keywords: Developmental Biology, embryo, Xenopus, organizer, animal pole, dissection
1Department of Cell Biology, Emory University
Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.
Published September 2, 2008. Keywords: Cellular Biology, Current Protocols Wiley, Xenopus Egg Extracts, Nuclear Assembly, Nuclear Membrane
1Department of Chemistry, University of Toronto, 2Department of Chemistry, University of Wisconsin, 3Department of Chemistry, Duke University
The assembly of a nearfield infrared microscope for imaging protein aggregates is described.
Published November 25, 2009. Keywords: Cellular Biology, nearfield imaging, infrared, amyloid, fibril, protein
1Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen
Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.
Published November 16, 2013. Keywords: Basic Protocols, FtsZ, protein polymerization, cell division, GTPase, sedimentation assay, light scattering
1Materials Engineering Division, Lawrence Livermore National Laboratory, 2UCSF Center for Integrative Neuroscience and the Department of Physiology, University of California, San Francisco
Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.
Published September 27, 2013. Keywords: Bioengineering, Nervous System Diseases, Surgical Procedures, Operative, Investigative Techniques, Nonmetallic Materials, Engineering (General), neural interfaces, polymer neural probes, surgical insertion, polyethylene glycol, microelectrode arrays, chronic implantation
1Center for Neutron Science, Department of Chemical and Biomolecular Engineering, University of Delaware, 2NIST Center for Neutron Research, National Institute of Standards and Technology, 3Institut Laue-Langevin
A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.
Published February 6, 2014. Keywords: Physics, Surfactants, Rheology, Shear Banding, Nanostructure, Neutron Scattering, Complex Fluids, Flow-induced Structure
1Institute of Laboratory Animal Science, University of Zurich, 2Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota
Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.
Published April 2, 2014. Keywords: Genetics, Oocyte microinjection, Designer nucleases, ZFN, TALEN, Genome Engineering
1Department of Biology, Case Western Reserve University
Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community’s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.
Published March 13, 2014. Keywords: Environmental Sciences, Coexistence, community assembly, environmental drivers, plant-soil feedback, soil heterogeneity, soil microbial communities, soil patch
1Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation
A protocol is described that uses Illumina's Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.
Published November 19, 2013. Keywords: Basic Protocol, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
1Institute for Critical Technology and Applied Science, Virginia Tech, 2Macromolecules and Interfaces Institute, Virginia Tech, 3Institute for Food Safety and Health, Illinois Institute of Technology- Moffett Campus, 4Wood, Cellulose, and Paper Research Department, University of Guadalajara, 5Department of Sustainable Biomaterials, Virginia Tech, 6Sustainable Nanotechnology Interdisciplinary Graduate Education Program, Virginia Tech
The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions. Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.
Published June 17, 2014. Keywords: Plant Biology, nanocellulose, thin films, quartz crystal microbalance, layer-by-layer, LbL
1Department of Molecular Genetics, The Ohio State University
Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard.
Published December 20, 2014. Keywords: Cellular Biology, Centrosome assembly, cell cycle, centrosomal degradation, quantitative fluorescence microscopy, normalization, VDAC3, BrdU pulse-chase
1Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, 2Department of Obstetrics, Gynecology & Reproductive Sciences, University of California, San Francisco, 3Keck School of Medicine, University of Southern California
We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.
Published June 27, 2014. Keywords: Bioengineering, liquid injection molding, reaction injection molding, molds, 3D printing, fused deposition modeling, rapid prototyping, medical devices, low cost, low volume, rapid turnaround time.
1Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535
Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.
Published May 21, 2014. Keywords: Cellular Biology, Stress granule (SG), G3BP, primary cells, neurons
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.
Published June 6, 2012. Keywords: Bioengineering, Biomedical Engineering, electrodeposition, biofabrication, chitosan, alginate, lab-on-a-chip, microfluidic, DTRA
1Department of Mechanical Engineering, University of Michigan
A procedure for studying transient flows near boundaries using high-resolution, high-speed particle image velocimetry (PIV) is described here. PIV is a non-intrusive measurement technique applicable to any optically accessible flow by optimizing several parameter constraints such as the image and recording properties, the laser sheet properties, and analysis algorithms.
Published June 24, 2013. Keywords: Physics, Mechanical Engineering, Fluid Mechanics, flow measurement, fluid heat transfer, internal flow in turbomachinery (applications), boundary layer flow (general), flow visualization (instrumentation), laser instruments (design and operation), Boundary layer, micro-PIV, optical laser diagnostics, internal combustion engines, flow, fluids, particle, velocimetry, visualization
1Center for Computational Medicine and Bioinformatics, University of Michigan, 2Center for Bioinformatics and Department of Molecular Bioscience, University of Kansas
Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.
Published November 3, 2011. Keywords: Biochemistry, On-line server, I-TASSER, protein structure prediction, function prediction
1Naval Research Laboratory
A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.
Published May 9, 2014. Keywords: Chemistry, phase transfer, nanoparticle, self-assembly, bottom-up, fabrication, low-cost, monolayer, thin film, nanostructure, array, metamaterial
1Institute of Photonics and Optical Sciences (IPOS), School of Physics, University of Sydney
Metamaterials at terahertz frequencies offer unique opportunities, but are challenging to fabricate in bulk. We adapt the fabrication procedure for microstructured polymer optical fibers to inexpensively fabricate metamaterials potentially on an industrial scale. We produce polymethylmethacrylate fibers containing ~10 μm diameter indium wires separated by ~100 μm, which exhibit a terahertz plasmonic response.
Published October 18, 2012. Keywords: Physics, Optics, Photonics, Materials Science, Fiber drawing, metamaterials, polymer optical fiber, microstructured fibers
1Organic Chemistry Institute and CeNTech, Westfälische Wilhelms-Universität Münster, 2Laboratory of Macromolecular and Organic Chemistry, Institute for Complex Molecular Systems, Eindhoven University of Technology, 3Laboratory of Materials and Interface Chemistry and Soft Matter Research Unit, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology
The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.
Published August 2, 2012. Keywords: Chemical Engineering, Chemistry, Physics, Self-assembly, cryogenic transmission electron microscopy, circular dichroism, controlled architecture, discotic amphiphile
1Department of Biology, Georgetown University, 2Insect Physiology Lab, EEOB, The Ohio State University
RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.
Published November 30, 2014. Keywords: Genetics, Aedes albopictus Asian tiger mosquito, photoperiodic diapause, RNA-Seq de novo transcriptome assembly, mosquito husbandry
1Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, 2Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory, 3Presently at the Interdisciplinary Center for Wide Band-gap Semiconductors, University Of California Santa Barbara
Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.
Published December 9, 2011. Keywords: Bioengineering, Direct-write assembly, silver ink, 3D printing, planar, three-dimensional, microelectrodes, flexible electronics, printed electronics
1Department of Psychiatry, University of Pittsburgh Medical Center, 2Department of Bioengineering, Stanford University, 3Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, 4Department of Neurobiology and Behavior, Cornell University, 5Department of Psychiatry and Behavioral Sciences, Stanford University
Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.
Published January 15, 2015. Keywords: Neuroscience, optogenetics, rodent, behavior, opsin, channelrhodopsin, brain, fiber optics, laser, neural circuits
1The Neuroscience Institute, New York University Langone Medical Center, 2Department of Brain and Cognitive Science, Massachusetts Institute of Technology
Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.
Published September 8, 2014. Keywords: Neuroscience, multi-electrode, micro-drives, electrophysiology, single units, brain circuit recording, deep brain structure
1Tissue Engineered Mechanics, Imaging and Materials Laboratory, Department of Biomedical Engineering, Florida International University, 2Department of Mechanical and Aerospace Engineering, University of Florida, 3College of Medicine, University of Florida, 4King Faisal Specialty Hospital and Research Center, Jeddah, Saudi Arabia
There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.
Published October 17, 2013. Keywords: Bioengineering, Cardiovascular Diseases, Circulatory and Respiratory Physiological Phenomena, Fluid Mechanics and Thermodynamics, Mechanical Engineering, valve disease, valve replacement, polymer valves, pulse duplicator, modification, tri-leaflet geometries, hydrodynamic studies, relative assessment, medicine, bioengineering, physiology
JoVE Immunology and Infection
1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, 2Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, NY
We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.
Published July 16, 2012. Keywords: Virology, Immunology, Molecular Biology, Genetics, cell fusion, HCV, restriction factor, heterokaryon, mouse, species-specificity
JoVE Developmental Biology
1Feinberg Cardiovascular Research Institute, Northwestern University, 2Cardiovascular Research Institute, University of California, San Francisco
Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.
Published March 26, 2015. Keywords: Developmental Biology, Immunofluorescence, mouse, embryonic heart, cardiomyocyte, development, sarcomere, intercalated disc, costamere, s-α-actinin, cryosection
1Department of Biomedical Engineering, Duke University, 2School of Medicine, Duke University, 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania
A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.
Published September 9, 2011. Keywords: Bioengineering, Stent, Titanium, Thrombosis, Endothelial Progenitor Cell, Endothelium, Biomaterial, Biocompatibility, Bioengineering, Translational Medicine, Vascular Surgery, Porcine
1Lab of Immune and Neural Networks, Feinstein Institute for Medical Research, North Shore LIJ Health System, 2Department of Molecular Medicine, Hofstra North Shore LIJ School of Medicine
The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.
Published July 5, 2013. Keywords: Behavior, Neuroscience, Neurobiology, Anatomy, Physiology, Biomedical Engineering, Brain, Amygdala, Hippocampus, Electrodes, Implanted, Microelectrodes, Action Potentials, Neurosciences, Neurophysiology, Neuroscience, brain, mouse, in vivo electrophysiology, tetrodes, microdrive, chronic recordings, local field potential, dorsal subiculum, animal model
1Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, 2Federal Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia, 3Physiology Department, Perelman School of Medicine, University of Pennsylvania
Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.
Published March 15, 2014. Keywords: Basic Protocol, microscopy flow chamber, single-molecule fluorescence, laser trap, microtubule-binding protein, microtubule-dependent motor, microtubule tip-tracking
1Center for Bio/Molecular Science & Engineering, US Naval Research Laboratory, 2Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill
Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.
Published January 8, 2014. Keywords: Bioengineering, hydrodynamic focusing, polymer fiber, biohybrid, microfabrication, sheath flow, click chemistry
1Biomedical Engineering Program, University of Delaware, 2Department of Materials Science and Engineering, Delaware Biotechnology Institute, University of Delaware
A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.
Published August 1, 2014. Keywords: Bioengineering, vocal fold; bioreactor; speaker; silicone membrane; fibrous scaffold; mesenchymal stem cells; vibration; extracellular matrix
1Department of Biology, California Institute of Technology, 2Department of Bioengineering, California Institute of Technology, 3Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, 4School of Physics and Astronomy, University of Minnesota
This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.
Published September 16, 2013. Keywords: Cellular Biology, Bioengineering, Synthetic Biology, Chemistry Techniques, Synthetic, Molecular Biology, control theory, TX-TL, cell-free expression, in vitro, transcription-translation, cell-free protein synthesis, synthetic biology, systems biology, Escherichia coli cell extract, biological circuits, biomolecular breadboard
1Department of Pharmacy and Biotechnology, University of Bologna, 2CSGI, Department of Chemistry, University of Firenze
Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.
Published February 5, 2015. Keywords: Biochemistry, Escherichia coli, protein co-expression, compatible plasmids, complementation test, DNA polymerase III, mutator strains