9780199638093

Zebrafish A Practical Approach

by ;
  • ISBN13:

    9780199638093

  • ISBN10:

    0199638098

  • Edition: 1st
  • Format: Hardcover
  • Copyright: 2002-11-21
  • Publisher: Oxford University Press
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Supplemental Materials

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  • The New copy of this book will include any supplemental materials advertised. Please check the title of the book to determine if it should include any access cards, study guides, lab manuals, CDs, etc.

Summary

The zebrafish has become one of the most important model organisms to study biologial processes in vivo. As a vertebrate that has many of the strengths of invertebrate model systems, it offers numerous advantages to researchers interested in many aspects of embryonic development, physiologyand disease. The next few years will see the completion of large scale initiatives that exploit the zebrafish as a model system for the understanding of gene function in vertebrates, including the sequencing of the genome. The zebrafish will therefore play an increasingly important role in the future ofbiomedical research. Whole genome sequencing projects, such as the human genome project, have led to the isolation of tens of thousands of genes for which the in vivo function is unknown. It is therefore likely that an increasing number of researchers will turn to organisms such as the zebrafish tounderstand the in vivo requirement for the proteins these genes encode.Recent technical advances now allow the rapid testing of in vivo function of as yet uncharacterised genes in zebrafish in large numbers and at a speed that is impossible in other systems. This book not only provides a complete set of instructions that will allow researchers to establish thezebrafish in their laboratory. It also gives a broad overview of commonly used methods and a comprehensive collection of protocols describing the most powerful techniques.

Table of Contents

List of protocols
xiii
Abbreviations xvii
Introduction: zebrafish as a system to study development and organogenesis 1(212)
Christiane Nusslein-Volhard
Darren T. Gilmour
Ralf Dahm
History
1(1)
Phylogeny
2(1)
The zebrafish genome
2(2)
Advantages of the zebrafish as a model system
4(1)
Purpose of this book
4(3)
Acknowledgements
5(1)
References
5(2)
Keeping and raising zebrafish
7(32)
Michael Brand
Michael Granato
Christiane Nusslein-Volhard
Introduction
7(1)
Aquaria systems and water conditions
7(5)
Aquaria systems
7(3)
Aquaria
10(2)
Water
12(3)
Fresh fish water
12(2)
Filtering
14(1)
Room conditions and maintenance
15(3)
Room conditions
15(1)
Temperature
16(1)
Illumination
16(1)
Cleaning
16(2)
Safety
18(1)
Fish care
18(10)
Keeping adult fish
18(1)
Raising fish
19(9)
Maintaining stocks
28(6)
Maintaining wild-type stocks
28(1)
Maintaining mutant stocks
28(1)
Identifying and maintaining adult heterozygous mutant carriers
29(1)
Maintaining mutant stocks as frozen sperm
30(4)
Fish health
34(5)
Fish diseases
34(1)
Fish handling in systems with latent TB
35(1)
Quarantine
36(1)
References
37(2)
Looking at embryos
39(20)
Stefan Schulte-Merker
Introduction
39(1)
In situ hybridization
39(6)
Single probe
40(3)
Detecting two differently labelled probes in situ
43(2)
Antibody staining
45(6)
Detecting antigens in early embryos using biotinylated secondary antibodies
45(2)
Antibody staining of zebrafish larvae
47(1)
Double in situ hybridization combined with antibody staining (triple stain)
48(2)
Pre-absorption of antibodies
50(1)
Mounting
51(2)
Viewing chambers for observing embryos
51(1)
Methyl cellulose mounting
51(1)
Araldite mounting
52(1)
Mounting in benzyl benzoate/benzyl alcohol
53(1)
Preparing sections
53(4)
Plastic sections using Technovit
54(1)
Paraffin sections combined with antibody staining
54(1)
Embedding and sectioning using JB-4 resin
55(1)
Toluidine Blue staining of semi-thin sections
56(1)
PTU treatment to prevent melanization of embryos
57(1)
Endothelial cell staining
57(2)
Acknowledgements
58(1)
References
58(1)
The morphology of larval and adult zebrafish
59(36)
Thomas F. Schilling
Introduction
59(1)
Body proportions and surface features
59(6)
Adult
59(2)
Larvae and juveniles
61(4)
Skeleton and musculature
65(11)
Adult skeleton
65(4)
Larval and juvenile skeletal development
69(4)
Adult musculature
73(1)
Larval and juvenile muscle development
74(2)
Nervous system
76(11)
Adult central nervous system
76(3)
Larval and juvenile neural development
79(4)
Adult peripheral nervous system and sense organs
83(3)
Larval and juvenile development of the PNS and sense organs
86(1)
Cardiovascular, digestive, and reproductive organs
87(8)
Adult
87(4)
Larval and juvenile organogenesis
91(1)
Acknowledgements
92(1)
References
92(3)
Cell labelling and transplantation techniques
95(26)
Donald A. Kane
Yasuyuki Kishimoto
Introduction
95(1)
Cell labelling techniques
96(7)
Labelling whole embryos with lineage tracer dyes
97(4)
Labelling individual blastomeres after 256-cell stage
101(1)
Labelling groups of cells by photo-activation
102(1)
Labelling groups of cells by lipophilic membrane dyes
103(1)
Transplantation techniques
103(7)
Precision transplantation of small groups of cells
104(4)
Large-scale transplantations of early blastomeres
108(1)
Transplantation of pieces of embryos
109(1)
Procedures for observing labelled cells
110(11)
Procedures for observing live material
111(3)
Procedures for preparing fixed material
114(3)
Procedures for analysing data
117(2)
References
119(2)
Manipulating gene expression in the zebrafish
121(24)
Darren T. Gilmour
Jason R. Jessen
Shuo Lin
Introduction
121(1)
Microinjection of zebrafish embryos
121(7)
Equipment required
122(3)
The microinjection procedure
125(3)
Transient expression approaches
128(9)
DNA constructs and mRNA transcripts give different expression profiles upon injection
129(1)
RNA injection
129(2)
DNA microinjection and its applications
131(1)
Promoter analysis
132(1)
Co-injection
133(1)
Rescue of mutants using transgenesis
134(1)
Artificial chromosome transgenesis in zebrafish
134(3)
Generation of stable germ line transgenic lines
137(4)
Identifying transgenic founder fish by PCR
138(1)
Identification of transgenic founder fish by reporter gene expression
139(1)
Generation of homozygous transgenic fish
140(1)
Morpholino `knockdown' of gene activity
141(4)
References
143(2)
Mutagenesis
145(30)
Francisco Pelegri
Introduction
145(1)
Mutagenic agents
145(7)
Chemical mutagens: N-ethyl-N-nitrosourea
145(4)
Radiation sources
149(1)
Insertional mutagenesis
150(2)
Selection of background genetic lines
152(1)
General criteria for selecting genetic backgrounds
152(1368)
Selection of lines amenable for in vitro fertilization and parthenogenesis
152(1)
Selection of lines free of lethal or sterile mutations
153(1)
Genetic screening strategies
153(19)
Inbreeding of families carrying non-mosaic germ lines
153(3)
Screens involving ploidy manipulation
156(11)
Screens for recessive mutations affecting adult traits and maternal-effect mutations
167(1)
Screens for dominant mutations
167(1)
Allele screens
167(5)
Screen tests
172(1)
Morphological screens
172(1)
Screens using molecular markers and tissue stains
172(1)
Locomotion and behavioural screens
172(1)
Conclusions
172(3)
Acknowledgements
173(1)
References
173(2)
Mapping and cloning
175(38)
Robert Geisler
Maps of the zebrafish genome
175(2)
Mapping mutations using SSLP markers
177(15)
Mapping approaches
177(1)
Selection of zebrafish lines and SSLPs
178(2)
Mapping strategy
180(3)
Set-up for high-throughput mapping
183(2)
Evaluating pooled PCR gels
185(1)
Evaluating single-embryo PCR gels
186(3)
Calculation of a map position
189(3)
Fine mapping
192(1)
Radiation hybrid mapping of candidate genes
192(3)
Matching mutations with candidate genes
195(4)
Searching maps and databases
195(1)
Evidence for the identity of a mutant locus
196(1)
Demonstrating a direct linkage by SNP genotyping
196(3)
Positional cloning of mutations
199(14)
Genomic libraries
199(2)
Screening for genomic clones
201(6)
Analysing a genomic clone
207(3)
Acknowledgements
210(1)
References
210(3)
Appendix 1 List of suppliers 213(6)
Appendix 2 Atlas of embryonic stages of development in the zebrafish 219(18)
Ralf Dahm
Appendix 3 Table of zebrafish mutations 237(56)
Hans Georg Frohnhofer
Index 293

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