Zebrafish : a practical approach / edited by Christiane Nüsslein-Volhard and Ralf Dahm.

Format:

Book

Contributor:

Nüsslein-Volhard, C. (Christiane)

Dahm, Ralf.

Series:

Practical approach series ; 261.

The practical approach series ; no. 261

Language:

English

Subjects (All):

Zebra danios as laboratory animals.

Animal models in research.

Zebrafish.

Models, Animal.

Animals, Laboratory.

Medical Subjects:

Zebrafish.

Models, Animal.

Animals, Laboratory.

Physical Description:

xviii, 303 pages, 8 unnumbered pages of plates : illustrations (some color) ; 26 cm.

Edition:

First edition.

Place of Publication:

Oxford ; New York : Oxford University Press, 2002.

Summary:

The zebrafish offers numerous advantages to researchers interested in embryonic development, physiology and disease. In the next few years the entire zebrafish genome will be sequenced, and several large scale initiatives that exploit the zebrafish as a model system for the understanding of gene function in vertebrates will be complete. The zebrafish will therefore play an increasingly important role in the future of biomedical research. Recent technical advances now enable rapid testing of the in vivo function of large numbers of uncharacterised genes in zebrafish, at speeds impossible in other vertebrate systems. This book provides a complete set of instructions to allow researchers to use zebrafish in their laboratory. It also provides a comprehensive collection of protocols for the most powerful techniques to study them. These include the analysis of embryos using in situ hybridization and antibody staining, cell labelling and transplantation methods, along with techniques for manipulating gene expression, mutagenesis, mapping and cloning. An atlas of embryonic stages of development in the zebrafish and an invaluable compilation of all published zebrafish mutations are also included. This book is an essential source of information and a unique laboratory manual for all researchers who currently work on zebrafish and those planning to do so.

Contents:

Introduction: zebrafish as a system to study development and organogenesis / Christiane Nusslein-Volhard, Darren T. Gilmour, Ralf Dahm 1

1 History 1

2 Phylogeny 2

3 The zebrafish genome 2

4 Advantages of the zebrafish as a model system 4

1 Keeping and raising zebrafish / Michael Brand, Michael Granato, Christiane Nusslein-Volhard 7

2 Aquaria systems and water conditions 7

3 Water 12

Fresh fish water 12

Filtering 14

4 Room conditions and maintenance 15

Temperature 16

Illumination 16

Cleaning 16

Safety 18

5 Fish care 18

Keeping adult fish 18

Raising fish 19

6 Maintaining stocks 28

Maintaining wild-type stocks 28

Maintaining mutant stocks 28

Identifying and maintaining adult heterozygous mutant carriers 29

Maintaining mutant stocks as frozen sperm 30

7 Fish health 34

Fish diseases 34

Fish handling in systems with latent TB 35

Quarantine 36

2 Looking at embryos / Stefan Schulte-Merker 39

2 In situ hybridization 39

Single probe 40

Detecting two differently labelled probes in situ 43

3 Antibody staining 45

Detecting antigens in early embryos using biotinylated secondary antibodies 45

Antibody staining of zebrafish larvae 47

Double in situ hybridization combined with antibody staining (triple stain) 48

Pre-absorption of antibodies 50

4 Mounting 51

Viewing chambers for observing embryos 51

Methyl cellulose mounting 51

Araldite mounting 52

Mounting in benzyl benzoate/benzyl alcohol 53

5 Preparing sections 53

Plastic sections using Technovit 54

Paraffin sections combined with antibody staining 54

Embedding and sectioning using JB-4 resin 55

Toluidine Blue staining of semi-thin sections 56

6 PTU treatment to prevent melanization of embryos 57

7 Endothelial cell staining 57

3 The morphology of larval and adult zebrafish / Thomas F. Schilling 59

2 Body proportions and surface features 59

Adult 59

Larvae and juveniles 61

3 Skeleton and musculature 65

Adult skeleton 65

Larval and juvenile skeletal development 69

Adult musculature 73

Larval and juvenile muscle development 74

4 Nervous system 76

Adult central nervous system 76

Larval and juvenile neural development 79

Adult peripheral nervous system and sense organs 83

Larval and juvenile development of the PNS and sense organs 86

5 Cardiovascular, digestive, and reproductive organs 87

Adult 87

Larval and juvenile organogenesis 91

4 Cell labelling and transplantation techniques / Donald A. Kane, Yasuyuki Kishimoto 95

2 Cell labelling techniques 96

Labelling whole embryos with lineage tracer dyes 97

Labelling individual blastomeres after 256-cell stage 101

Labelling groups of cells by photo-activation 102

Labelling groups of cells by lipophilic membrane dyes 103

3 Transplantation techniques 103

Precision transplantation of small groups of cells 104

Large-scale transplantations of early blastomeres 108

Transplantation of pieces of embryos 109

4 Procedures for observing labelled cells 110

Procedures for observing live material 111

Procedures for preparing fixed material 114

Procedures for analysing data 117

5 Manipulating gene expression in the zebrafish / Darren T. Gilmour, Jason R. Jessen, Shuo Lin 121

2 Microinjection of zebrafish embryos 121

Equipment required 122

The microinjection procedure 125

3 Transient expression approaches 128

DNA constructs and mRNA transcripts give different expression profiles upon injection 129

RNA injection 129

DNA microinjection and its applications 131

Promoter analysis 132

Co-injection 133

Rescue of mutants using transgenesis 134

Artificial chromosome transgenesis in zebrafish 134

4 Generation of stable germ line transgenic lines 137

Identifying transgenic founder fish by PCR 138

Identification of transgenic founder fish by reporter gene expression 139

Generation of homozygous transgenic fish 140

5 Morpholino 'knockdown' of gene activity 141

6 Mutagenesis / Francisco Pelegri 145

2 Mutagenic agents 145

Chemical mutagens: N-ethyl-N-nitrosourea 145

Radiation sources 149

Insertional mutagenesis 150

3 Selection of background genetic lines 152

General criteria for selecting genetic backgrounds 152

Selection of lines amenable for in vitro fertilization and parthenogenesis 1520

Selection of lines free of lethal or sterile mutations 153

4 Genetic screening strategies 153

Inbreeding of families carrying non-mosaic germ lines 153

Screens involving ploidy manipulation 156

Screens for recessive mutations affecting adult traits and maternal-effect mutations 167

Screens for dominant mutations 167

Allele screens 167

5 Screen tests 172

Morphological screens 172

Screens using molecular markers and tissue stains 172

Locomotion and behavioural screens 172

7 Mapping and cloning / Robert Geisler 175

1 Maps of the zebrafish genome 175

2 Mapping mutations using SSLP markers 177

Mapping approaches 177

Selection of zebrafish lines and SSLPs 178

Mapping strategy 180

Set-up for high-throughput mapping 183

Evaluating pooled PCR gels 185

Evaluating single-embryo PCR gels 186

Calculation of a map position 189

Fine mapping 192

3 Radiation hybrid mapping of candidate genes 192

4 Matching mutations with candidate genes 195

Searching maps and databases 195

Evidence for the identity of a mutant locus 196

Demonstrating a direct linkage by SNP genotyping 196

5 Positional cloning of mutations 199

Genomic libraries 199

Screening for genomic clones 201

Analysing a genomic clone 207

Appendix 2 Atlas of embryonic stages of development in the zebrafish / Ralf Dahm 219

Appendix 3 Table of zebrafish mutations / Hans Georg Frohnhofer 237.

Notes:

Includes bibliographical references and index.

ISBN:

0199638098

019963808X

OCLC:

49926870