1NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health
Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.
Published July 24, 2014. Keywords: Stem Cell Biology, Pluripotent stem cells, human embryonic stem cells, induced pluripotent stem cells, cell culture, non-colony type monolayer, single cell, plating efficiency, Rho-associated kinase, Y-27632, transfection, transduction
1Program in Epithelial Biology, Stanford University School of Medicine
Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.
Published February 28, 2013. Keywords: Genetics, Tissue Engineering, Medicine, Biomedical Engineering, Cellular Biology, Surgery, Epithelial Biology, regeneration, chamber, hair, follicle, dermis, dermal cells, keratinocyte, graft, epithelial, cell culture, lentivirus, knockdown, shRNA-mediated knockdown, overexpression, mice, transgenic mice, animal model
1Boyce Thompson Institute for Plant Research, Cornell University
Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.
Published August 12, 2009. Keywords: Plant Biology, mating, zygote, tetrad, mating type, Chlamydomonas
JoVE Immunology and Infection
1Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth
Here we report a method for using type I and type II Δku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.
Published July 12, 2013. Keywords: Infectious Diseases, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
1Department of Molecular Genetics, University of Toronto, 2Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 3Department of Biochemistry, Research and Innovation Centre, University of Regina
Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.
Published November 12, 2012. Keywords: Genetics, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
1Donald Danforth Plant Science Center, 2Boyce Thompson Institute
We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.
Published October 1, 2013. Keywords: Environmental Sciences, Hybridization, Genetics, plants, Setaria viridis, crosses, emasculation, flowering, seed propagation, seed dormancy
1Department of Biological Sciences, University of Notre Dame
The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.
Published July 14, 2014. Keywords: Developmental Biology, zebrafish, haploid, in vitro fertilization, forward genetic screen, saturation, recessive mutation, mutagenesis
JoVE Clinical and Translational Medicine
1Department of Biology, Western Kentucky University
Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.
Published September 11, 2013. Keywords: Medicine, Imaginal Discs, Drosophila melanogaster, Neoplasm Metastasis, Drosophila, Invasive Tumors, Benign Tumors, Cephalic Complex, Mosaic Analysis with a Repressible Cell Marker technique
1Institute for Cell and Molecular Biosciences, Newcastle University Medical School
Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.
Published August 13, 2012. Keywords: Physiology, Medicine, Robotic, microbial, culture, yeast, array, library, high-throughput, analysis, fitness, growth rate, quantitative, solid agar
1Biotechnology and Cellular Signalling, UMR 7242 CNRS, Université de Strasbourg
We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of naïve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.
Published July 29, 2014. Keywords: Neuroscience, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
JoVE Immunology and Infection
1Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center
We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.
Published October 23, 2013. Keywords: Immunology, genetics, chemical mutagenesis, whole genome sequencing
1Department of Molecular and Integrative Physiology, University of Michigan, 2Cellular and Molecular Biology Program, University of Michigan
Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.
Published January 7, 2013. Keywords: Developmental Biology, Cellular Biology, Molecular Biology, Anatomy, Physiology, Entomology, longevity, lifespan, aging, Drosophila melanogaster, fruit fly, Drosophila, mortality, animal model
1Neuroscience Center of Excellence, Louisiana State University Health Sciences Center
Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.
Published December 5, 2013. Keywords: Biochemistry, Drosophila, GAL4/UAS system, transgenic, Tandem Affinity Purification, protein-protein interaction, proteomics
JoVE Clinical and Translational Medicine
1Department of Exercise Science, Elon University, 2Department of Physical Therapy Education, Elon University, 3Department of Physical Therapy, Duquesne University, 4Department of Sports Medicine, Elon University
There is great variability in an individual’s risk for concussion and their corresponding recovery. A multifaceted approach to concussion evaluation is warranted; including baseline testing of athletes before participation in sport and timely evaluation post injury. The goal of this protocol is to provide an appropriate multifaceted approach to examine concussions.
Published December 8, 2014. Keywords: Medicine, Concussions, Student-Athletes, Mild Traumatic Brain Injury, Genetics, Cognitive Function, Balance, Gait, Somatosensory
1Department of Biological and Allied Health Sciences, Fairleigh Dickinson University
An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.
Published October 22, 2013. Keywords: Developmental Biology, Microbiology, C. elegans, Behavior, Animal, Microbiology, Caenorhabditis elegans, Enterococcus faecalis, egg-laying behavior, animal model
JoVE Immunology and Infection
1Department of Biology, Boston College
Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.
Published February 8, 2012. Keywords: Immunology, Genetics, Toxoplasma gondii, chemical mutagenesis, egress, genetic screen
1Department of Human Genetics, University of Utah
Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.
Published July 9, 2013. Keywords: Developmental Biology, Genetics, Molecular Biology, Cellular Biology, Biochemistry, Biophysics, Bioengineering, Cellular Structures, Epithelial Cells, Drosophila melanogaster, Microscopy, Phase-Contrast Microscopy, Fluorescence Microscopy, genetics (animal and plant), animal biology, animal models, Respiratory System, trachea, terminal cell, intact animal, larvae, cell morphology, Drosophila, fluorescence, branching, lumen, fruit fly, animal model
1RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research
High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.
Published May 31, 2013. Keywords: Genetics, Molecular Biology, Biochemistry, Virology, Cancer Biology, Medicine, Genomics, Nucleic Acid Probes, RNA Probes, RNA, High-throughput SHAPE, Capillary electrophoresis, RNA structure, RNA probing, RNA folding, secondary structure, DNA, nucleic acids, electropherogram, synthesis, transcription, high throughput, sequencing
1Center for Applied Proteomics and Molecular Medicine, George Mason University, 2Virginia Surgery Associates
Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for >6 months.
Published November 8, 2014. Keywords: Cancer Biology, Breast, ductal carcinoma in situ, epidermal growth factor, mammosphere, organoid, pre-invasive, primary cell culture, serum-free, spheroid
1School of Molecular Biosciences, Washington State University, 2Center for Reproductive Biology, Washington State University
Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.
Published November 29, 2013. Keywords: Biochemistry, Caenorhabditis elegans, C. elegans, Nutrition Therapy, genetics (animal and plant), Polyunsaturated fatty acids, omega-6, omega-3, dietary fat, dihomo-gamma-linolenic acid, germ cells
1Department of Molecular and Cellular Biology, Harvard University
The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.
Published March 19, 2012. Keywords: Cellular Biology, Caenorhabditis elegans, extracellular fluid, reverse microinjection, vampiric isolation, pseudocoelom
1Institute of Genetics, University of Mainz, 2Department of Anatomy and Neuroscience, University of Melbourne
We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.
Published March 4, 2013. Keywords: Developmental Biology, Neuroscience, Neurobiology, Genetics, Cellular Biology, Molecular Biology, Anatomy, Drosophila, fruit fly, Neurosciences, Neuroanatomy, Life sciences, embryonic nervous system, central nervous system, neuronal morphology, single cell labeling, embryo, microscopy, animal model
JoVE Clinical and Translational Medicine
1The Markey Cancer Center, University of Kentucky College of Medicine, 2Graduate Center for Toxicology, University of Kentucky College of Medicine, 3Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, 4Department of Pediatrics, University of Kentucky College of Medicine
Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.
Published September 7, 2013. Keywords: Medicine, Skin, Inflammation, Photometry, Ultraviolet Rays, Skin Pigmentation, melanocortin 1 receptor, Mc1r, forskolin, cAMP, mean erythematous dose, skin pigmentation, melanocyte, melanin, sunburn, UV, inflammation
1Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill
We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.
Published February 20, 2013. Keywords: Biochemistry, Molecular Biology, Anatomy, Physiology, Basic Protocols, Surgery, Zebrafish, Danio rerio, intestine, lumen, larvae, gavage, microgavage, epithelium, barrier function, gut motility, microsurgery, microscopy, animal model
1Department of Cell and Developmental Biology, Vanderbilt University Medical Center
Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.
Published January 20, 2014. Keywords: Basic Protocol, Drosophila melanogaster, dissection, testes, spermatogenesis, meiosis, germ cells, phase-contrast microscopy, immunofluorescence
1Department of Physiology and Biophysics, Boston University School of Medicine, 2Boston University Photonics Center
With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.
Published April 10, 2013. Keywords: Developmental Biology, Physiology, Biophysics, Neurobiology, Cellular Biology, Molecular Biology, Anatomy, Developmental Biology, Biomedical Engineering, Medicine, Caenorhabditis elegans, C. elegans, Microscopy, Fluorescence, Neurosciences, calcium imaging, genetically encoded calcium indicators, cameleon, GCaMP, neuronal activity, time-lapse imaging, laser ablation, optical neurophysiology, neurophysiology, neurons, animal model
JoVE Immunology and Infection
1Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio, 2Department of Cellular and Structural Biology, UT Health Science Center at San Antonio, 3Department of Pathology, UT Health Science Center at San Antonio, 4Department of Microbiology, UT Health Science Center at San Antonio, 5Cancer Therapy and Research Center, UT Health Science Center at San Antonio
Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model. The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants spontaneously arisen or after exposure to genotoxins.
Published February 24, 2014. Keywords: Infection, In vivo mutagenesis, hematopoietic stem/progenitor cells, LacI mouse model, DNA mutations, E. coli
JoVE Immunology and Infection
1Center for Molecular Bacteriology and Infection, Imperial College London
The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.
Published November 22, 2013. Keywords: Infection, Bacterial Infections, Infection, Disease Models, Animal, Bacterial Infections and Mycoses, Galleria mellonella, Legionella pneumophila, insect model, bacterial infection, Legionnaires' disease, haemocytes
1Department of Biological Sciences, University of Toledo
Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.
Published September 20, 2013. Keywords: Developmental Biology, biology (general), genetics (animal and plant), animal biology, animal models, Life Sciences (General), Centrosome, Spermatogenesis, Spermiogenesis, Drosophila, Centriole, Cilium, Mitosis, Meiosis
1Neuroscience Graduate Group, University of Pennsylvania School of Medicine, 2Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine
Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.
Published July 27, 2011. Keywords: Cell Biology, Yeast, plasmid, transformation, PEG/LioAc, high-throughput screen
1Department of Genetics, Albert Einstein College of Medicine, 2Shanghai Key Laboratory of Complex Prescription and MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine
Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.
Published November 15, 2013. Keywords: Biochemistry, Orphan nuclear receptor, ketoconazole, yeast two-hybrid, Pregnane X Receptor, ligand, antatogist, coactivators SRC-1 (steroid receptor coactivator 1), drug-receptor interaction
1Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 2Department of Microbial and Environmental Genomics, J. Craig Venter Institute, 3Donnelly Centre & Department of Molecular Genetics, University of Toronto, 4Lunenfeld Research Institute, Mt Sinai Hospital
The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.
Published December 15, 2012. Keywords: Microbiology, Genetics, Synthetic Biology, Environmental Genomics, Genomics, Bioengineering, Biomedical Engineering, Cellular Biology, Multi-site genomic engineering, genetic interaction, green fluorescent protein, GFP, flow cytometry, Saccharomyces cerevisiae, yeast, Green Monster
1Department of Biology and Centre for Species at Risk and Habitat Studies,, University of British Columbia, Okanagan Campus
We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.
Published March 13, 2011. Keywords: Genetics, Conservation genetics, noninvasive genetic sampling, Hair snares, Microsatellites, AFLPs, American pika, Ochotona princeps
1Department of Biological Sciences, Florida Atlantic University, 2Department of Chemistry & Biochemistry, Florida Atlantic University
A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.
Published April 15, 2012. Keywords: Neuroscience, Drosophila melanogaster, Giant Fiber Circuit, screening, in vivo, nanoinjection, electrophysiology, modulatory compounds, biochemistry
1Department of Biology, New York University
The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.
Published November 14, 2012. Keywords: Neuroscience, Anatomy, Physiology, Immunology, Developmental Biology, Drosophila, retina, photoreceptor, imaginal disc, larva, pupa, confocal microscopy, immunohistochemistry
1Pacific Northwest Diabetes Research Institute
Barcode Enabled Sequencing of Tetrads (BEST) replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by fluorescence-activated cell sorting onto agar plates, separates the spores by agitation with glass beads, and determines which randomly arrayed colonies were derived from the same original tetrad using molecular barcodes.
Published May 1, 2014. Keywords: Genetics, Yeast, Tetrad, Genetics, DNA sequencing
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.
Published March 26, 2011. Keywords: Genetics, DNA extraction, paraffin embedded tissue, phenol:chloroform extraction, genetic analysis, epigenetic analysis
1The Visual Systems Group, Division of Pediatric Ophthalmology, Cincinnati Childrens Hospital Medical Center, 2PRESTO, JST
We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here we describe the design and analysis of TPR in Drosophila.
Published January 13, 2014. Keywords: Basic Protocol, Drosophila, circadian clock, temperature, temperature preference rhythm, locomotor activity, body temperature rhythms
1Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, 2Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST), 3Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, National Institutes of Natural Sciences
This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.
Published March 1, 2014. Keywords: Behavior, Fear, Learning, Memory, ImageFZ program, Mouse, contextual fear, cued fear
1Department of Biology, Institute of Cell and Developmental Biology, University of Fribourg
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.
Published April 20, 2013. Keywords: Neuroscience, Developmental Biology, Neurobiology, Behavior, Molecular Biology, Cellular Biology, Physiology, Anatomy, Light, preference test, Drosophila, larva, fruit fly, visual behavior, circadian rhythm, visual system, animal model, assay
1Department of Crop Sciences, University of Illinois Urbana-Champaign
Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.
Published September 4, 2014. Keywords: Neuroscience, C. elegans, dauer, dendrite, arborization, phenotypic plasticity, stress, imaging, pheromone
JoVE Immunology and Infection
1Biology Department, The City College of New York, CUNY, 2The Graduate Center, The City University of New York
Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.
Published May 7, 2012. Keywords: Immunology, Parasitoid wasps, innate immunity, encapsulation, hematopoiesis, insect, fat body, Toll-NF-kappaB, molecular biology
JoVE Immunology and Infection
1Department of Integrative Biology, University of California, Berkeley
The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage: sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.
Published March 21, 2014. Keywords: Immunology, Innate Immunity, C. elegans, Pseudomonas aeruginosa, wormsorter, pathogen recognition
1Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania
Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.
Published November 11, 2014. Keywords: Microbiology, Protein-misfolding disorders, yeast proteinopathy models, Hsp104, proteotoxicity, amyloid, disaggregation
1Language and Genetics Department, Max Planck Institute for Psycholinguistics, 2Donders Institute for Brain, Cognition and Behaviour
Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.
Published May 26, 2014. Keywords: Cellular Biology, Protein-protein interactions, Bioluminescence Resonance Energy Transfer, Live cell, Transfection, Luciferase, Yellow Fluorescent Protein, Mutations
1Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Donnelly Sequencing Centre, University of Toronto, 4Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, 5Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, 6Department of Pharmaceutical Sciences, University of Toronto
We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.
Published August 11, 2011. Keywords: Biochemistry, chemical biology, chemogenomics, chemical probes, barcode microarray, next generation sequencing
1Centre d’Immunologie de Marseille-Luminy, Université de la Méditerranée
We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.
Published February 27, 2012. Keywords: Molecular Biology, C. elegans, fluorescent reporter, Biosort, LIMS, innate immunity, Drechmeria coniospora
1Department of Pathology, University of Washington, 2Molecular and Cellular Biology Program, University of Washington
In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.
Published May 12, 2009. Keywords: Developmental Biology, Caenorhabditis elegans, aging, longevity, life span assay, worms, nematode, dietary restriction, RNA interference
1Department of Biology, University of Kentucky, Lexington
We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.
Published November 16, 2009. Keywords: Cellular Biology, Invertebrate, myocyte, pacemaker, insect
JoVE Immunology and Infection
1Department of Entomology, Virginia Tech
Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).
Published January 6, 2014. Keywords: Immunology, Microdissection, whole genome amplification, malaria mosquito, polytene chromosome, mitotic chromosomes, fluorescence in situ hybridization, chromosome painting