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A collection of standard and cutting-edge techniques for using Xenopus oocytes and oocytes/egg extracts to reconstitute biological and cellular processes. These readily reproducible methods take advantage of the oocyte's impressive protein abundance, its striking protein translation capacity, and its breathtaking possibilities for the assembly of infectious viral particles by single cell injection of multiple RNAs. The authors focus on the versatility of frog oocytes and egg extracts in cell biology and signal transduction, and cover all the major uses of oocytes/extracts as experimental models. Although Xenopus oocytes are very hardy and robust in terms of experimentation and offer unusually simple and inexpensive opportunities for the delivery of mRNA to the cytoplasm and DNA to the nucleus, they are underutilized as an experimental model. In Xenopus Protocols: Cell Biology and Signal Transduction, hands-on researchers describe standard and cutting-edge techniques for using Xenopus oocytes and oocyte/egg extracts to reconstitute biological and cellular processes. These readily reproducible methods take advantage of the oocyte's impressive protein abundance (for example, 50-70 ng catalytic subunit of PKA per oocyte), its striking protein translation capacity (200-400 ng protein/oocyte per day), and its breathtaking possibilities for the assembly of infectious viral particles by single-cell injection of multiple RNAs. The authors focus on the versatility of frog oocytes and egg extracts in cell biology and signal transduction and cover all the major uses of oocytes/extracts as experimental models. The protocols follow the successful Methods in Molecular Biology series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Comprehensive and highly practical, Xenopus Protocols: Cell Biology and Signal Transduction serves perfectly to instruct novices in the use of Xenopus oocytes/extracts, as well as seasoned investigators interested in developing novel models and uses.
List of contents
Resources for Genetic and Genomic Studies of Xenopus.- The Physiology of the Xenopus laevis Ovary.- Oocyte Isolation and Enucleation.- Xenopus tropicalis Oocytes.- Oocyte Expression With Injection of Purified T7 RNA Polymerase.- Visualization of the Cytoskeleton in Xenopus Oocytes and Eggs by Confocal Immunofluorescence Microscopy.- Multiphoton Laser Scanning Microscopy as a Tool for Xenopus Oocyte Research.- Imaging Ca2+ Signals in Xenopus Oocytes.- Chromosomal DNA Replication in a Soluble Cell-Free System Derived From Xenopus Eggs.- Chromatin Assembly of DNA Templates Microinjected Into Xenopus Oocytes.- Pre-mRNA Splicing in the Nuclei of Xenopus Oocytes.- Chromatin Immunoprecipitation for Studying Transcriptional Regulation in Xenopus Oocytes and Tadpoles.- Cytoplasmic mRNA Polyadenylation and Translation Assays.- Xenopus Egg Extracts.- Analysis of Molecular Chaperones Using a Xenopus Oocyte Protein Refolding Assay.- Ubiquitin-Mediated Protein Degradation in Xenopus Egg Extracts.- to Nucleocytoplasmic Transport.- Use of Xenopus laevis Oocyte Nuclei and Nuclear Envelopes in Nucleocytoplasmic Transport Studies.- Nuclear Pore Complex Structure and Plasticity Revealed by Electron and Atomic Force Microscopy.- In Vitro Study of Nuclear Assembly and Nuclear Import Using Xenopus Egg Extracts.- Use of Intact Xenopus Oocytes in Nucleocytoplasmic Transport Studies.- Studying the Mechanosensitivity of Voltage-Gated Channels Using Oocyte Patches.- Oocytes as an Expression System for Studying Receptor/Channel Targets of Drugs and Pesticides.- Microtransplantation of Neurotransmitter Receptors From Cells to Xenopus Oocyte Membranes.- Reconstitution of Golgi Disassembly by Mitotic Xenopus Egg Extract in Semi-Intact MDCK Cells.- Exploring RNA Virus Replication in Xenopus Oocytes.- Study of Apoptosis In Vitro Using the Xenopus Egg Extract Reconstitution System.- Studying Fertilization in Cell-Free Extracts.- Localized Sampling, Electrophoresis, and Biosensor Analysis of Xenopus laevis Cytoplasm for Subcellular Biochemical Assays.- Monitoring Protein Kinase A Activities Using Expressed Substrate in Live Cells.- Using Xenopus Oocyte Extracts to Study Signal Transduction.- Oocyte Extracts for the Study of Meiotic M-M Transition.- Methods for Studying Spindle Assembly and Chromosome Condensation in Xenopus Egg Extracts.
Summary
A collection of standard and cutting-edge techniques for using Xenopus oocytes and oocytes/egg extracts to reconstitute biological and cellular processes. These readily reproducible methods take advantage of the oocyte's impressive protein abundance, its striking protein translation capacity, and its breathtaking possibilities for the assembly of infectious viral particles by single cell injection of multiple RNAs. The authors focus on the versatility of frog oocytes and egg extracts in cell biology and signal transduction, and cover all the major uses of oocytes/extracts as experimental models.
