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Xenopus: A Laboratory Manual

Subject Area(s):  Microscopy and ImagingDevelopmental BiologyCell BiologyMolecular BiologyXenopus laevisBiochemistryCytology

Edited by Hazel L. Sive, Northeastern University

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© 2023 • 1,067 pages, illustrated (142 color and 18 B&W ), index
Paperback • $165 132.00
ISBN  978-1-621821-80-9
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  •     Description    
  •     Contents    


Members of the frog Xenopus genus are versatile model organisms used in developmental biology, cell biology, neuroscience, and toxicology research, among others. Frogs are easy to rear and maintain, and large sample sizes of embryos and later stages are readily obtained. Studies using Xenopus cell-free extracts, oocytes, eggs, embryos, larval stages, and adult frogs have yielded important insights into a multitude of key biological processes—from mechanisms underlying the cell cycle to embryonic development to human disease.

This new laboratory manual provides a comprehensive collection of experimental procedures for research using Xenopus. It includes basic methods for Xenopus husbandry, egg harvesting, embryo collection, microdissection, and visualization, as well as strategies for extract preparation, genetic manipulation, genomic analysis, transcriptomics, and proteomics. There are protocols for analyses of Xenopus immunology, neurobiology, metamorphosis, and adult physiology. Toxicological studies and chemical screening methods are also included.

The manual covers both Xenopus laevis and the smaller Xenopus tropicalis species. It is a critical reference for laboratories that currently work with these organisms and will serve as an essential start-up guide for those seeking to start using Xenopus frogs in their research for the first time.


Edward M. De Robertis and John B. Gurdon
Hazel L. Sive
Editors: Richard M. Harland and Hazel L. Sive
Xenopus Husbandry
Richard M. Harland and Hazel L. Sive
1 Animal Maintenance Systems: Xenopus laevis
Nikko-Ideen Shaidani, Sean McNamara, Marcin Wlizla, and Marko E. Horb
2 Animal Maintenance Systems: Xenopus tropicalis
Nikko-Ideen Shaidani, Sean McNamara, Marcin Wlizla, and Marko E. Horb
3 Raising and Maintaining Xenopus tropicalis from Tadpole to Adult
aura Lane, Michael Slocum, and Mustafa K. Khokha
4 Defining the Specific Pathogen-Free State of Xenopus Using TaqMan Assays
Casey L. Hensley, Kaitlin M. Bowes, and Sanford H. Feldman
SECTION 2Eggs to Adult
Editors: Hazel L. Sive and Richard M. Harland
Obtaining Xenopus Eggs and Embryos
Hazel L. Sive and Richard M. Harland
1 Obtaining Xenopus laevis Eggs
Nikko-Ideen Shaidani, Sean McNamara, Marcin Wlizla, and Marko E. Horb
2 Obtaining Xenopus laevis Embryos
Nikko-Ideen Shaidani, Sean McNamara, Marcin Wlizla, and Marko E. Horb
3 How to Grow Xenopus laevis Tadpole Stages to Adult
Shoko Ishibashi and Enrique Amaya
4 Obtaining Xenopus tropicalis Eggs
Maura Lane, Emily K. Mis, and Mustafa K. Khokha
5 Obtaining Xenopus tropicalis Embryos by In Vitro Fertilization
Maura Lane and Mustafa K. Khokha
6 Obtaining Xenopus tropicalis Embryos by Natural Mating
Maura Lane and Mustafa K. Khokha
7 Microinjection of Xenopus tropicalis Embryos
Maura Lane, Emily K. Mis, and Mustafa K. Khokha
8 Best Practices for Xenopus tropicalis Husbandry
Takuya Nakayama and Robert M. Grainger
SECTION 3Oocytes and Oogenesis
Editor: Kimberly L. Mowry
Using the Xenopus Oocyte Toolbox
Kimberly L. Mowry
1 Isolation of Xenopus Oocytes
Karen Newman, Tristan Aguero, and Mary Lou King
2 Isolation and Analysis of Xenopus Germinal Vesicles
Garry T. Morgan
3 Microinjection of Xenopus Oocytes
Tristan Aguero, Karen Newman, and Mary Lou King
4 Oocyte Host-Transfer and Maternal mRNA Depletion Experiments in Xenopus 105
Douglas W. Houston
5 Heterologous Protein Expression in the Xenopus Oocyte
Jonathan S. Marchant
6 Patch-Clamp and Perfusion Techniques to Study Ion Channels Expressed in Xenopus Oocytes
Guohui Zhang and Jianmin Cui
7 Whole-Mount In Situ Hybridization of Xenopus Oocytes
Diana Bauermeister and Tomas Pieler
8 Whole-Mount Immunofluorescence for Visualizing Endogenous Protein and Injected RNA in Xenopus Oocytes
Samantha P. Jeschonek and Kimberly L. Mowry
9 Fluorescence In Situ Hybridization of Cryosectioned Xenopus Oocytes
Christopher R. Neil and Kimberly L. Mowry
SECTION 4Embryology I
Editor: Sally A. Moody
Analysis of Cell Fate Commitment in Xenopus Embryos
Sally A. Moody
1 Lineage Tracing and Fate Mapping in Xenopus Embryos
Sally A. Moody
2 Microinjection of mRNAs and Oligonucleotides
Sally A. Moody
3 Microinjection of DNA Constructs into Xenopus Embryos for Gene Misexpression and cis-Regulatory Module Analysis
Yuuri Yasuoka and Masanori Taira
4 Whole-Mount In Situ Hybridization of Xenopus Embryos
Jean-Pierre Saint-Jeannet
5 Whole-Mount Immunocytochemistry in Xenopus
Michael W. Klymkowsky
6 Cleavage Blastomere Explant Culture in Xenopus
Sally A. Moody
7 Cleavage Blastomere Deletion and Transplantation to Test Cell Fate Commitment in Xenopus
Sally A. Moody
8 Dissecting and Culturing Animal Cap Explants
Kevin S. Dingwell and James C. Smith
SECTION 5Embryology II
Editor: Sally A. Moody
Xenopus Explants and Transplants
Sally A. Moody
1 Spemann-Mangold Grafts
Hélène Cousin
2 Einsteck Transplants
Hélène Cousin
3 Microsurgical Methods to Isolate and Culture the Early Gastrula Dorsal Marginal Zone
Lance A. Davidson
4 Microsurgical Manipulations to Isolate Collectively Migrating Mesendoderm
Lance A. Davidson
5 Microsurgical Methods to Make the Keller Sandwich Explant and the Dorsal Isolate
Lance A. Davidson
6 Cranial Neural Crest Explants
Hélène Cousin and Dominique Alfandari
7 Cranial Neural Crest Transplants
Hélène Cousin
8 In Vitro Induction of Xenopus Embryonic Organs Using Animal Cap Cells
Takashi Ariizumi, Tatsuo Michiue, and Makoto Asashima
9 Special Considerations for Making Explants and Transplants with Xenopus tropicalis
Marilyn Fisher and Robert M. Grainger
SECTION 6Cell Biology
Editor: Anna Philpott
The Use of Xenopus for Cell Biology Applications
Anna Philpott
1 In Vivo Assessment of Neural Precursor Cell Cycle Kinetics in the Amphibian Retina
Morgane Locker and Muriel Perron
2 Applying Tensile and Compressive Force to Xenopus Animal Cap Tissue
Georgina K. Goddard, Nawseen Tarannum, and Sarah Woolner
3 Analysis of Phosphorylation Status of Ectopically Expressed Proteins in Early Xenopus Embryos
Laura J.A. Hardwick and Anna Philpott
4 Analysis of Chromatin Binding of Ectopically Expressed Proteins in Early Xenopus Embryos
Laura J.A. Hardwick and Anna Philpott
5 Raising Antibodies for Use in Xenopus
Maya Z. Piccinni and Matthew J. Guille
6 Assessing the Immune Response When Raising Antibodies for Use in Xenopus
Maya Z. Piccinni and Matthew J. Guille
7 Confirming Antibody Specificity in Xenopus
Sian A. Martin, Suzannah J. Page, Maya Z. Piccinni, and Matthew J. Guille
8 Purifying Antibodies Raised against Xenopus Peptides
Maya Z. Piccinni and Matthew J. Guille
SECTION 7Xenopus Extract Systems
Editor: Rebecca Heald
The Use of Cell-Free Xenopus Extracts to Investigate Cytoplasmic Events
Romain Gibeaux and Rebecca Heald
1 Preparation of Cellular Extracts from Xenopus Eggs and Embryos
Matthew C. Good and Rebecca Heald
2 Robustly Cycling Xenopus laevis Cell-Free Extracts in Teflon Chambers
Jeremy B. Chang and James E. Ferrell, Jr.
3 Protein Immunodepletion and Complementation in Xenopus laevis Egg Extracts
Christopher Jenness, David J. Wynne, and Hironori Funabiki
4 Assembly of Spindles and Asters in Xenopus Egg Extracts
Christine M. Field and Timothy J. Mitchison
5 Dissecting Protein Complexes in Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts
Jae-Geun Song and Sabine Petry
6 Filopodia-Like Structure Formation from Xenopus Egg Extracts
Helen M. Fox and Jennifer L. Gallop
7 Endoplasmic Reticulum Network Formation with Xenopus Egg Extracts
Songyu Wang, Fabian B. Romano, and Tom A. Rapoport
8 Microfluidic Encapsulation of Demembranated Sperm Nuclei in Xenopus Egg Extracts
John Oakey and Jesse C. Gatlin
9 Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems
Gary S. McDowell and Anna Philpott
10 Assessing Ubiquitylation of Individual Proteins Using Xenopus Extract Systems
Gary S. McDowell and Anna Philpott
SECTION 8Chromosome Dynamics In Vitro
Editor: Rebecca Heald
Reconstituting Nuclear and Chromosome Dynamics Using Xenopus Extracts
Susannah Rankin
1 Isolation and Demembranation of Xenopus Sperm Nuclei
James W. Hazel and Jesse C. Gatlin
2 Chromosome Cohesion and Condensation in Xenopus Egg Extracts
Eulália M.L. da Silva and Susannah Rankin
3 Chromatin Characterization in Xenopus laevis Cell-Free Egg Extracts and Embryos
Wei-Lin Wang, Takashi Onikubo, and David Shechter
4 Centromere and Kinetochore Assembly in Xenopus laevis Egg Extract
Julio C. Flores Servin and Aaron F. Straight
5 Analysis of Mitotic Checkpoint Function in Xenopus Egg Extracts
Yinghui Mao
6 Nucleus Assembly and Import in Xenopus laevis Egg Extract
Pan Chen and Daniel L. Levy
7 Extracts for Analysis of DNA Replication in a Nucleus-Free System
Justin Sparks and Johannes C. Walter
SECTION 9Imaging
Editors: Lance A. Davidson and Laura Anne Lowery
Imaging Methods in Xenopus Cells, Embryos, and Tadpoles
Lance A. Davidson and Laura Anne Lowery
1 Whole-Mount RNA In Situ Hybridization and Immunofluorescence of Xenopus Embryos and Tadpoles
Helen Rankin Willsey
2 Cryosectioning and Immunostaining of Xenopus Embryonic Tissues
Olga Ossipova and Sergei Y. Sokol
3 Live Imaging of Cytoskeletal Dynamics in Embryonic Xenopus laevis Growth Cones and Neural Crest Cells
Burcu Erdogan, Elizabeth A. Bearce, and Laura Anne Lowery
4 Xenopus Tadpole Craniocardiac Imaging Using Optical Coherence Tomography
Engin Deniz, Emily K. Mis, Maura Lane, and Mustafa K. Khokha
5 Chambers for Culturing and Immobilizing Xenopus Embryos and Organotypic Explants for Live Imaging
Chih-Wen Chu and Lance A. Davidson
SECTION 10Genetics and Gene Editing
Editors: Matthew J. Guille and Robert M. Grainger
Genetics and Gene Editing Methods in Xenopus laevis and Xenopus tropicalis
Matthew J. Guille and Robert M. Grainger
1 CRISPR-Cas9 Mutagenesis in Xenopus tropicalis for Phenotypic Analyses in the F0 Generation and Beyond
Ira L. Blitz and Takuya Nakayama
2 Generating Nonmosaic Mutants in Xenopus Using CRISPR-Cas in Oocytes
Sang-Wook Cha
3 Tissue-Targeted CRISPR-Cas9-Mediated Genome Editing of Multiple Homeologs in F0-Generation Xenopus laevis Embryos
Mark E. Corkins, Bridget D. DeLay, and Rachel K. Miller
4 Modeling Human Genetic Disorders with CRISPR Technologies in Xenopus
Helen Rankin Willsey, Matthew J. Guille, and Robert M. Grainger
5 Production of Transgenic F0 Animals and Permanent Lines by Sperm Nuclear Transplantation in Xenopus tropicalis
Takuya Nakayama, Jessica Gray, and Robert M. Grainger
6 I-SceI-Mediated Transgenesis in Xenopus
Anna Noble, Anita Abu-Daya, and Matthew J. Guille
7 Cryopreservation of Xenopus Sperm and In Vitro Fertilization Using Frozen Sperm Samples
Anna Noble, Anita Abu-Daya, and Matthew J. Guille
8 Homology-Directed Repair by CRISPR-Cas9 Mutagenesis in Xenopus Using Long Single-Stranded Donor DNA Templates via Simple Microinjection of Embryos
Takuya Nakayama, Robert M. Grainger, and Sang-Wook Cha
9 Gynogenetic Production of Embryos in Xenopus tropicalis Using a Cold Shock Procedure: Rapid Screening Method for Gene Editing Phenotypes
Takuya Nakayama, Amanda Cox, Mary Howell, and Robert M. Grainger
10 Preparation of Intact Nuclei for Single-Nucleus Omics Using Frozen Cell Suspensions from Mutant Embryos of Xenopus tropicalis
Takuya Nakayama, Janou A.Y. Roubroeks, Gert Jan C. Veenstra, and Robert M. Grainger
SECTION 11Genomics
Editor: Michael J. Gilchrist
Genomics Methods for Xenopus Embryos and Tissues
Michael J. Gilchrist, Ken W.Y. Cho, and Gert Jan C. Veenstra
1 ChIP-Sequencing in Xenopus Embryos
Saartje Hontelez, Ila van Kruijsbergen, and Gert Jan C. Veenstra
2 Assay for Transposase-Accessible Chromatin-Sequencing Using Xenopus Embryos
Ann Rose Bright and Gert Jan C. Veenstra
3 DNase-seq to Study Chromatin Accessibility in Early Xenopus tropicalis Embryos
Jin Sun Cho, Ira L. Blitz, and Ken W.Y. Cho
4 Generating a Three-Dimensional Genome from Xenopus with Hi-C
Ian K. Quigley and Sven Heinz
5 Mapping Chromatin Features of Xenopus Embryos
George E. Gentsch and James C. Smith
SECTION 12Transcriptomics and Proteomics
Editor: Michael J. Gilchrist
Transcriptomics and Proteomics Methods for Xenopus Embryos and Tissues
Michael J. Gilchrist, Gert Jan C. Veenstra, and Ken W.Y. Cho
1 An RNA-Seq Protocol for Differential Expression Analysis
Nick D.L. Owens, Elena De Domenico, and Michael J. Gilchrist
2 Mass Spectrometry-Based Absolute Quantification of Single Xenopus Embryo Proteomes
Rik G.H. Lindeboom, Arne H. Smits, Matteo Perino, Gert Jan C. Veenstra, and Michiel Vermeulen
3 INTACT Proteomics in Xenopus
Lauren Wasson, Nirav M. Amin, and Frank L. Conlon
SECTION 13Metamorphosis
Editors: Daniel R. Buchholz and Yun-Bo Shi
Methods for Investigating the Larval Period and Metamorphosis in Xenopus
Daniel R. Buchholz and Yun-Bo Shi
1 Xenopus Tadpole Tissue Harvest
Matthew D. Patmann, Leena H. Shewade, Katelin A. Schneider, and Daniel R. Buchholz
2 Organ Culture of the Xenopus Tadpole Intestine
Atsuko Ishizuya-Oka
3 Bulk Electroporation-Mediated Gene Transfer into Xenopus Tadpole Brain
Cristina Sáenz de Miera, Ethan Parr, and Robert J. Denver
4 In Vivo Transfection of Naked DNA into Xenopus Tadpole Tail Muscle
Lindsey Marshall, Fabrice Girardot, Barbara A. Demeneix, and Laurent Coen
5 Cell Proliferation Analysis during Xenopus Metamorphosis: Using 5-Ethynyl-2’-Deoxyuridine (EdU) to Stain Proliferating Intestinal Cells
Morihiro Okada and Yun-Bo Shi
6 Chromatin Immunoprecipitation for Chromatin Interaction Analysis Using Paired-End-Tag (ChIA-PET) Sequencing in Tadpole Tissues
Nicolas Buisine, Xiaoan Ruan, Yijun Ruan, and Laurent M. Sachs
7 Chromatin Interaction Analysis Using Paired-End-Tag (ChIA-PET) Sequencing in Tadpole Tissues
Nicolas Buisine, Xiaoan Ruan, Yijun Ruan, and Laurent M. Sachs
SECTION 14Neurobiology: Imaging
Editor: Hollis T. Cline
Imaging Structural and Functional Dynamics in Xenopus Neurons
Hollis T. Cline
1 In Vivo Time-Lapse Imaging and Analysis of Dendritic Structural Plasticity in Xenopus laevis Tadpoles
Hai-yan He, Chih-Yang Lin, and Hollis T. Cline
2 Imaging Mitochondrial Dynamics in the Xenopus Central Nervous System (CNS)
Martin Sihan Feng and Jennifer E. Bestman
3 Whole-Brain Calcium Imaging in Larval Xenopus
Thomas Offner, Daniela Daume, Lukas Weiss, Thomas Hassenklöver, and Ivan Manzini
4 Imaging the Dynamic Branching and Synaptic Differentiation of Xenopus Optic Axons In Vivo
Rommel Andrew Santos, Rodrigo Del Rio, Jr., and Susana Cohen-Cory
5 Bulk Dye Loading for In Vivo Calcium Imaging of Visual Responses in Populations of Xenopus Tectal Neurons
Peter W. Hogg and Kurt Haas
SECTION 15Neurobiology: Physiology and Behavior
Editors: Ben G. Szaro and Hollis T. Cline
Neurophysiological and Behavioral Analysis in Xenopus
Ben G. Szaro
1 Electrophysiological Recording for Study of Xenopus Retinotectal Circuitry
Yuhao Luo, Wanhua Shen, and Hollis T. Cline
2 Tetrode Recording in the Xenopus laevis Visual System Using Multichannel Glass Electrodes
Masaki Hiramoto and Hollis T. Cline
3 Electrophysiological Approaches to Studying Normal and Abnormal Retinotectal Circuit Development in the Xenopus Tadpole
Kara G. Pratt
4 Making In Situ Whole-Cell Patch-Clamp Recordings from Xenopus laevis Tadpole Neurons
Wen-Chang Li
5 Analysis of Visual Collision Avoidance in Xenopus Tadpoles
Arseny S. Khakhalin
6 Schooling in Xenopus laevis Tadpoles as a Way to Assess Their Neural Development
Virgilio Lopez III, Arseny S. Khakhalin, and Carlos Aizenman
7 Ex Vivo Brain Preparation to Analyze Vocal Pathways of Xenopus Frogs
Ayako Yamaguchi
SECTION 16Immunology
Editor: Jacques Robert
Experimental Platform Using the Amphibian Xenopus laevis for Research in Fundamental and Medical Immunology
Jacques Robert
1 Skin Grafting in Xenopus laevis: A Technique for Assessing Development and Immunological Disparity
Yumi Izutsu
2 Collagen-Embedded Tumor Transplantations in Xenopus laevis Tadpoles
Maureen Banach and Jacques Robert
3 Adoptive Transfer of Fluorescently Labeled Immune Cells in Xenopus
Kun Hyoe Rhoo and Jacques Robert
4 Flow Cytometric Analysis of Xenopus Immune Cells
Eva-Stina Edholm
5 Larval Thymectomy of Xenopus laevis
Sara Mashoof, Breanna Breaux, and Michael F. Criscitiello
6 Lymphocyte Deficiency Induced by Sublethal Irradiation in Xenopus
Louise A. Rollins-Smith and Jacques Robert
7 RNAi-Mediated Loss of Function of Xenopus Immune Genes by Transgenesis
Eva-Stina Edholm and Jacques Robert
8 Elicitation of Xenopus laevis Tadpole and Adult Frog Peritoneal Leukocytes
Leon Grayfer
9 Assessing Antibody Responses to Pathogens or Model Antigens in Xenopus by Enzyme-Linked Immunosorbent Assay (ELISA)
Francisco De Jesús Andino and Jacques Robert
SECTION 17Regeneration
Editor: Juan Larraín
Xenopus, a Model to Study Wound Healing and Regeneration: Experimental Approaches
Paula G. Slater, Miriam Palacios, and Juan Larraín
1 Studies of Limb Regeneration in Larval Xenopus
Anthony L. Mescher and Anton W. Neff
2 Cell Transplantation as a Method to Investigate Spinal Cord Regeneration in Regenerative and Nonregenerative Xenopus Stages
Emilio E. Méndez-Olivos and Juan Larraín
3 Methods for Examining Lens Regeneration in Xenopus
Jonathan J. Henry, Kimberly J. Perry, and Paul W. Hamilton
4 Ex Vivo Eye Tissue Culture Methods for Xenopus
Jonathan J. Henry, Kimberly J. Perry, and Paul W. Hamilton
5 Investigating the Cellular and Molecular Mechanisms of Wound Healing in Xenopus Oocytes and Embryos
Jingjing Li and Enrique Amaya
6 Tracing Central Nervous System Axon Regeneration in Xenopus
Kurt M. Gibbs and Ben G. Szaro
7 Rod-Specific Ablation Using the Nitroreductase/Metronidazole System to Investigate Regeneration in Xenopus
Reyna I. Martinez-De Luna and Michael E. Zuber
8 Infrared Laser-Mediated Gene Induction at the Single-Cell Level in the Regenerating Tail of Xenopus laevis Tadpoles
Riho Hasugata, Shinichi Hayashi, Aiko Kawasumi-Kita, Joe Sakamoto, Yasuhiro Kamei, and Hitoshi Yokoyama
SECTION 18Chemical Screening and Toxicity Testing
Editor: André W. Brändli
Chemical Screening and Toxicity Testing
André W. Brändli
1 Chemical Screening Using Cell-Free Xenopus Egg Extract
Matthew R. Broadus and Ethan Lee
2 Screening of Chemical Libraries Using Xenopus Embryos and Tadpoles for Phenotypic Drug Discovery
Mazhar Gull, Stefan M. Schmitt, Roland E. Kälin, and André W. Brändli
3 Inverse Drug Screening of Bioelectric Signaling and Neurotransmitter Roles: Illustrated Using a Xenopus Tail Regeneration Assay
Kelly G. Sullivan and Michael Levin
4 Following Endocrine-Disrupting Effects on Gene Expression in Xenopus laevis
Petra Spirhanzlova, Michelle Leemans, Barbara A. Demeneix, and Jean-Baptiste Fini
5 In Vivo Assessment of Drug-Induced Hepatotoxicity Using Xenopus Embryos
Katy Saide and Grant N. Wheeler
6 Frog Embryo Teratogenesis Assay—Xenopus (FETAX): Use in Alternative Preclinical Safety Assessment
Douglas J. Fort and Michael Mathis