Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.
Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.
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.
Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos
The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.
Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction – sperm accumulation assays and sperm tracking assays.
Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation
This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.
Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.
The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.
The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.
The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.
Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.
Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures
This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.
Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.
Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.
Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.
Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.
IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation
1Center for Biological Security, Robert Koch-Institute, 2CICVyA - INTA Castelar, Instituto de Virología, 3Center of Molecular Immunology, Ciudad de la Habana, Cuba, 4Department of Biology, Chemistry, Pharmacy, Institute of Biology-Neurobiology, Free University of Berlin, 5Institut of Pharmacology, Charité-University Medicine of Berlin
This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.
Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling
We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.
Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.
Procedure for fertilizing Xenopus oocytes by the host transfer method.
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.
Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.
Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.
To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.
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.
Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.
Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.
The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human β-amyloid peptide (Aβ). Induced expression of Aβ in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate Aβ toxicity.
The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.
This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.
To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.
The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.
The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.
Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.
This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.
Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice
1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research
A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.
Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.
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.
Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.
A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.
Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.
Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.
In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.
A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.
This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.
1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University
Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.
Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.
Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.
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.
We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.