Xenopus Protocols: Post-Genomic Approaches

556 Pages·2012·9.636 MB·English

by  Robert M. Grainger (auth.), STEFAN HOPPLER, Peter D Vize (eds.)

M M B ™
ETHODS IN OLECULAR IOLOGY
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John M. Walker
School of Life Sciences
University of Hertfordshire
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Xenopus Protocols
Post-Genomic Approaches
Second Edition
Edited by
Stefan Hoppler
Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK
Peter D. Vize
Department of Biological Science, University of Calgary, Calgary, Canada
Editors
Stefan Hoppler Peter D. Vize
Institute of Medical Sciences Department of Biological Science
University of Aberdeen University of Calgary
Foresterhill, Aberdeen, UK Calgary, Canada
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-61779-991-4 ISBN 978-1-61779-992-1 (eBook)
DOI 10.1007/978-1-61779-992-1
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Preface
The Xenopus system had a long and prominent history during the twentieth century as an
accessible model system for studying vertebrate biology. Many discoveries in biochemistry,
cell biology, and in particular in developmental biology have their roots in experimental
investigation using X enopus . Among the inherent advantages of the Xenopus model are the
ease with which large numbers of eggs can be collected; the large size of those eggs and the
embryos that develop from them after fertilization; a consistent fate map; the external
development of these embryos and the speed with which the conserved vertebrate body
plan is organized. The large cells in the early embryos were ideal for addressing cell biologi-
cal questions but also allowed early embryonic patterning of the vertebrate embryo and the
fate map to be unraveled. Many fundamental processes in vertebrate development were fi rst
discovered in X enopus in areas such as axial development, mesoderm induction, neural pat-
terning, and more recently organogenesis and regeneration, to name but a few. The absence
of well-developed genetics has often been cited as the one disadvantage of the X enopus
laevis model system compared to mouse or zebrafi sh for instance, but as this volume shows
very clearly this criticism has been thoroughly put to rest.
This volume of Methods in Molecular Biology on Xenopus comes at a crucial juncture
for our model system. The recent publication of the draft genome for Xenopus tropicalis
marks an important milestone. It reveals that the X enopus genome is in itself an ideal model
for vertebrate genomes since it manifests conserved vertebrate genomic organization and
therefore reinforces the uniquely advantageous phylogenetic position of X enopus as a gen-
eral vertebrate model. The genome and the embrace of genomic approaches enabled by
X. tropicalis as an experimental model have invigorated the entire X enopus fi eld, not just
those working with X. tropicalis . This volume seeks to focus on these new approaches.
X. tropicalis offers tractable genetics that will complement the traditional strengths of
Xenopus as a model system, as do the powerful transgenics methods pioneered in Xenopus .
The genome and new deep sequencing approaches open up a new chapter in the analysis of
gene expression on a whole transcriptome level and of protein expression and interaction
on a proteome level; here in particular the traditional advantages of the X enopus model
system synergize very directly with the new information technology available to enable
completely novel approaches. However, this impact is also felt in new methodology to
image these expressed transcripts and proteins in embryos and in cells, particularly with
sophisticated fl uorescence microscopy; and of course in new systems approaches and appli-
cations for stem cell technology and regeneration. As a consequence, the computer and
online databases will become as much a tool for X enopus researchers in the future as the
micropipette, the injection needle, and the dissection scalpel have been for many years.
v
vi Preface
The new approaches inspired by the X . tropicalis genome combined with the unique
experimental advantages of X . laevis and their distinctive phylogenetic position make them
unique model systems for vertebrate embryonic development in the twenty-fi rst century.
The protocols in this volume written by the leading experts in each method provide a tool-
kit that will enable every laboratory to maximize the power of this extraordinary experi-
mental system.
Aberdeen, UK Stefan Hoppler
Calgary, Canada Peter D. Vize
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
PART I XENOPUS TROPICALIS
1 Xenopus tropicalis as a Model Organism for Genetics
and Genomics: Past, Present, and Future. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Robert M. Grainger
2 Husbandry of Xenopus tropicalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Alan Jafkins, Anita Abu-Daya, Anna Noble,
Lyle B. Zimmerman, and Matthew Guille
3 Generating Diploid Embryos from Xenopus tropicalis . . . . . . . . . . . . . . . . . . . 33
Florencia del Viso and Mustafa Khokha
4 Navigating the Xenopus tropicalis Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Ira L. Blitz
PART II GENETICS IN XENOPUS
5 Genetic Analysis of Xenopus tropicalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Timothy J. Geach, Derek L. Stemple, and Lyle B. Zimmerman
6 Forward Genetic Screens in Xenopus Using Transposon-Mediated
Insertional Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Donald A. Yergeau, Clair M. Kelley, Haiqing Zhu,
Emin Kuliyev, and Paul E. Mead
7 Targeted Gene Disruption with Engineered Zinc-Finger
Nucleases (ZFNs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
John J. Young and Richard M. Harland
8 Reverse Genetic Studies Using Antisense Morpholino Oligonucleotides . . . . . 143
Yanan Zhao, Shoko Ishibashi, and Enrique Amaya
9 Chemical Genetics and Drug Discovery in Xenopus. . . . . . . . . . . . . . . . . . . . . 155
Matthew L. Tomlinson, Adam E. Hendry, and Grant N. Wheeler
10 Maternal mRNA Knock-down Studies: Antisense Experiments
Using the Host-Transfer Technique in Xenopus laevis
and Xenopus tropicalis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
David J. Olson, Alissa M. Hulstrand, and Douglas W. Houston
vii
viii Contents
PART III XENOPUS TRANGENESIS
11 Generating Transgenic Frog Embryos by Restriction Enzyme
Mediated Integration (REMI). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Shoko Ishibashi, Kristen L. Kroll, and Enrique Amaya
12 A Simple Method of Transgenesis using I-SceI
Meganuclease in Xenopus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Shoko Ishibashi, Nick R. Love, and Enrique Amaya
13 Using FC31 Integrase to Mediate Insertion of DNA
in Xenopus Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
You E. Li, Bryan G. Allen, and Daniel L. Weeks
14 Xenopus Transgenics: Methods Using Transposons . . . . . . . . . . . . . . . . . . . . . 231
Clair M. Kelley, Donald A. Yergeau, Haiqing Zhu,
Emin Kuliyev, and Paul E. Mead
15 Comparative Genomics-Based Identification and Analysis
of Cis-Regulatory Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Hajime Ogino, Haruki Ochi, Chihiro Uchiyama,
Sarah Louie, and Robert M. Grainger
16 Tet-On Binary Systems for Tissue-Specific and Inducible
Transgene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Daniel R. Buchholz
PART IV GENE EXPRESSION: FROM GENE TO TRANSCRIPT TO PROTEIN
17 Chromatin Immunoprecipitation Analysis of Xenopus Embryos. . . . . . . . . . . . 279
Robert C. Akkers, Ulrike G. Jacobi, and Gert Jan C. Veenstra
18 Transcriptomics Using Next Generation Sequencing Technologies . . . . . . . . . 293
Dasfne Lee-Liu, Leonardo I. Almonacid, Fernando Faunes,
Francisco Melo, and Juan Larrain
19 Databases of Gene Expression in Xenopus Development . . . . . . . . . . . . . . . . . 319
Michael J. Gilchrist and Nicolas Pollet
20 Investigating Alternative RNA Splicing in Xenopus . . . . . . . . . . . . . . . . . . . . . 347
Agnès Mereau and Serge Hardy
21 Immunoisolation of Protein Complexes from Xenopus. . . . . . . . . . . . . . . . . . . 369
Frank L. Conlon, Yana Miteva, Erin Kaltenbrun,
Lauren Waldron, Todd M. Greco, and Ileana M. Cristea
22 Complementary Proteomic Analysis of Protein Complexes . . . . . . . . . . . . . . . 391
Todd M. Greco, Yana Miteva, Frank L. Conlon,
and Ileana M. Cristea
PART V IMAGING XENOPUS DEVELOPMENT
23 Antibody Development and Use in Chromogenic
and Fluorescent Immunostaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Eamon Dubaissi, Niki Panagiotaki, Nancy Papalopulu,
and Peter D. Vize
Contents ix
24 Multiple Fluorescent In Situ mRNA Hybridization (FISH)
on Whole Mounts and Sections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Robert Lea, Boyan Bonev, Eamon Dubaissi, Peter D. Vize,
and Nancy Papalopulu
25 Methods to Analyze microRNA Expression and Function
During Xenopus Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Boyan Bonev and Nancy Papalopulu
26 A Bromodeoxyuridine (BrdU) Based Protocol for Characterizing
Proliferating Progenitors in Xenopus Embryos. . . . . . . . . . . . . . . . . . . . . . . . . 461
Hélène Auger, Raphaël Thuret, Warif El Yakoubi,
and Nancy Papalopulu
27 Microscopy Tools for Quantifying Developmental Dynamics
in Xenopus Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
Sagar D. Joshi, Hye Young Kim, and Lance A. Davidson
PART VI NOVEL APPROACHES IN XENOPUS
28 Mathematical Modeling of Gene Regulatory Networks
in Xenopus Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
Yasushi Saka
29 Stem-Cell-Like Embryonic Explants to Study Cardiac Development . . . . . . . . 515
Boni A. Afouda
30 Studying Regeneration in Xenopus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
Caroline W. Beck
31 On-Line Resources for Xenopus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
Jeff Bowes
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563