Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica
An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.
This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.
Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation
Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.
Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.
Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)
We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.
Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.
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.
1Microbiology and Pathogenesis Activity, Bacterial Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 2Tick-Borne Diseases Activity, Bacterial Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention
The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.
Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism
Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.
A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.
Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.
An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica
We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.
Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines
A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.
Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.
Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.
Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans
Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.
Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the December 2012 Issue of Journal of Visualized Experiments (JoVE).
A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.
Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.
In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.
Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans
1Department of Science, Borough of Manhattan Community College, City Universtiy of New York (CUNY), 2Department of Biology, Queens College, The City University of New York (CUNY), 3Biochemistry Program, The Graduate Center, Queens College, The City University of New York (CUNY)
We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-β signaling.
Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling
1Department of Biomedical Engineering, University of Virginia, 2Department of Biomedical Engineering, California Polytechnic State University, 3Office of Animal Welfare, University of Virginia, 4Department of Biomedical Engineering & Institute for Computational Medicine, Johns Hopkins University
We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.
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.
In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.
The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.
1Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute at Lake Nona, 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 3Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 4Department of Pediatrics and Cellular and Integrative Physiology, Indiana University School of Medicine
The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.
Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.
This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.
Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila
Morphological scaling relationships capture and describe organismal shape. We present a method to measure morphological scaling relationships across the natural range of body sizes in fully metamorphic insects. Using a simple diet manipulation we increase the distribution of trait sizes, permitting the accurate description of how shape and size co-vary.
In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals
A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.
We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.
Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes
This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.
1Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, 2Department of Biology, North Carolina Central University, Durham, 3Department of Physiology & Pharmacology and Hypertension & Vascular Research Center, Wake Forest University School of Medicine
An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently.
Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).
A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.
Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression
We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.
In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.
Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster
The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.
In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGCα1-/- mice using pressure myography.
Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.
Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.
Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.
This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.
Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.
We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.
A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo
Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.
Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.
1Stony Brook Children's Hospital, State University of New York at Stony Brook, 2Department of Pediatrics, State University of New York at Stony Brook, 3Department of Molecular Genetics, State University of New York at Stony Brook, 4Department of Microbiology, State University of New York at Stony Brook
We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.