Methods in Cell Biology

VOLUME 7 7

The Zebrafish : Genetics, Genomics, and Informatics

H. William Detrich, III

Monte Westerfield

Leonard I . Zon

Contributors

xv

Preface

Dedication

-cv

PART I Forward and Reverse Genetics

1 . Retroviral-Mediated Insertional Mutagenesis in Zebrafish

Adam Amsterdam and Nancy Hopkins

I. Introduction

3

II. Mutagenesis

5

III. Cloning the Mutated Genes

8

IV. Future Directions

1 4

References

1 8

2 . Genetic Screens for Maternal-Effect Mutations

Francisco Pelegri and Mary C . Mullins

I. Introduction

22

II. Strategies for Maternal-Effect Screens

23

III. Selection of Lines for Genetic Screens

3 3

IV. Recovery and Maintenance of Maternal-Effect Mutations

3 7

V. Mapping Maternal-Effect Mutations

4 1

VI. Solutions, Materials, and Protocols

44

VII. Conclusions

48

References

49

3 . Behavioral Screening Assays in Zebrafis h

Michael B . Orger, Ethan Gahtan, Akira Muto, Patrick Page-McCaw, Matthew C . Smear,

and Herwig Baier

I . Introduction

53

IL General Considerations

54

III. Behavioral Assays

5 6IV. Conclusions

6 6

References

6 6

4 . Target-Selected Gene Inactivation in Zebrafish

Erno Wienholds and Ronald H . A . Plasterk

I. Introduction

7 0II. Gene Targeting Strategies

7 1III. Target-Selected Mutagenesis in Zebrafish

7 5IV. Discussion

83V. Materials and Methods

8 5

References

87

5 . A High-Throughput Method for Identifying N-Ethyl-N-Nitrosourea (ENU)-Induce dPoint Mutations in Zebrafish

Bruce W. Draper, Claire M . McCallum, Jennifer L . Stout, Ann J. Slade, and Cecilia B . Moens

I. Introduction

92II. Rationale for Reverse Genetics in Zebrafish

95III. Rationale for Using the CEL1 Assay to Detect Induced Mutations

9 6IV. Rationale for Generating a Cryopreserved Mutant Library

9 8V. Method of N-Ethyl-N-Nitrosourea (ENU) Mutagenesis and Rearing of F l

Founder Fish

9 8VI. Generating a Cryopreserved Mutant Library

99VII. Isolating Genomic DNA

102VIII. Choosing Fragments to Screen

104IX. CEL1 Endonuclease Assay

104X. Recovery of Mutations from Cryopreserved Sperm

109XI. Materials Cost Estimate

11 0

References

11 1

6 . Production of Zebrafish Germline Chimeras by Using Cultured Embryoni cStern (ES) Cells

Lianchun Fan, Jennifer Crodian, and Paul Collodi

I. Introduction

11 3II. Methods

11 4III. Reagents

11 7IV. Future Directions

11 8

References

118

7 . Nonconventional Antisense in Zebrafish for Functional Genomics Applications

Stephen C . Ekker

I. Introduction

12 1

II. General Use of Nonconventional Antisense Tools in Zebrafish

12 3

III. Use of Morpholinos (MOs) as Antisense Tools in Zebrafish

12 5

IV. Comparison of MO-Based Screening Success to Mutational Methods

13 3

References

13 4

8 . Downregulation of Gene Expression with Negatively Charged Peptide Nucleic Acid s

(PNAs) in Zebrafish Embryo s

Eric Wickstrom, Karen A . Urtishak, Michael Choob, Xiaobing Tian, Nitzan Sternheim ,

Laura M . Cross, Amy Rubinstein, and Steven A . Farber

I. Introduction

13 8

II. Materials and Methods

143

III. Results

14 6

IV. Discussion

15 5

V. Summary

15 6

References

15 6

9 . Photo-Mediated Gene Activation by Using Caged mRNA in Zebrafish Embryo s

Hideki Ando, Toshiaki Furuta, and Hitoshi Okamot o

I. Introduction

160

II. Synthesis of Bhc-Caged mRNA

16 1

III. Microinjection of the Bhc-Caged mRNA

165

IV. Uncaging by Illumination of UV Light

167

V. Titration of Caging Efficiency

168

VI. Injection of Dimethyl Sulfoxide (DMSO) Solution of Caged mRNA

170

References

170

10 . Current Status of Medaka Genetics and Genomic sThe Medaka Genome Initiative (MGI)

Manfred Schartl, Indrajit Nanda, Manko Kondo, Michael Schmid, Shuichi Asakawa ,

Takashi Sasaki, Nobuyoshi Shimizu, Thorsten Henrich, Joachim Wittbrodt ,

Makoto Furutani-Seiki, Hisato Kondoh, Heinz Himmelbauer, Yunhan Hong,

Akihiko Koga, Masaru Nonaka, Hiroshi Mitani, and Akihiro Shim a

I. Introduction

17 4II. Current Status of Medaka Genetics

17 5

III. Current Status of Medaka Genomics

18 6

IV. Other Progress

193

V. Internet Web Sites Relevant to Medaka Resources and Databases

193References

194

11 . Transgenesis and Gene Trap Methods in Zebrafish by Using the To12Transposable Elemen t

Koichi Kawakami

I. Introduction

20 1II. Transgenesis by Using the To12 Transposable Element in Zebrafish

20 3III. A Gene Trap Approach that Uses the To12 Transposon System in Zebrafish 21 0IV. Summary and Perspectives

21 9References

22 0

PART II The Zebrafish Genome and Mapping Technologie s

12 . The Zebrafish Genome Project : Sequence Analysis and Annotation

Kerstin Jekosch

I. Introduction

22 5II. Automated Annotation of Whole Genome Shotgun (WGS) Assemblies

22 7III. Clone-by-Clone Analysis and Annotation

23 0IV. The Sanger Zebrafish Web Services

23 4V. Future Releases

23 8References

23 8

13 . Molecular Cytogenetic Methodologies and a Bacterial Artificial Chromosome (BAC )Probe Panel Resource for Genomic Analyses in Zebrafish

Charles Lee and Amanda Smith

I. Introduction

24 1II. Methods

24 2III. First-Generation Zebrafish BAC Probe Panel

24 8References

25 4

14 . Automated Analysis of Conserved Syntenies for the Zebrafish Genom e

John Postlethwait, Victor Ruotti, Michael J . Carvan, and Peter" Tonellato

I. Introduction

25 6II. The Conservation of Zebrafish and Mammalian Genomes

25 6III. Using Genome Conservation in Zebrafish Research

258

IV. Genomic Connections Between Zebrafish and Tetrapods Facilitat eDisease Research

260

V. Bioinformatic Approaches to Automating Zebrafish Connectionsand Comparative Maps

264

VI. A Conserved Syntenic Map for Zebrafish : Preliminary Results

266

VII. Conclusion

267References

268

15 . Update of the Expressed Sequence Tag (EST) and Radiation Hybrid Panel Projects

Yi Zho u

I. Expressed Sequence Tag (EST) Projects

27 4

II. Radiation Hybrid (RH) Panel Project

277

III. Future Directions

29 1

References

29 2

16 . Bacterial Artificial Chromosome (BAC) Clones and the Current Clone Map of the

Zebrafish Genome

Romke Koch, Gerd-Jörg Rauch, Sean Humphray, Robert Geisler, and Ronald Plasterk

I. Introduction

29 6

II. Physical Map of Fingerprinted Clones

29 6

III. Screening Methods and Utilization of the Current Zebrafish Clone Map

300

IV. Example : Discovery of the Zebrafish dicerl Gene

302

References

304

17 . The Zon Laboratory Guide tö Positional Cloning in Zebrafis h

Nathan Bahary, Alan Davidson, David Ransom, Jennifer Shepard, Howard Stern ,

Nikolaus Trede, Yi Zhou, Bruce Barut, and Leonard I . Zo n

I. Introduction

30 6

II. Mapping Strains

30 6

III. Families and Genetic Markers

307

IV. Crosses for Line Maintenance and Mapping

307

V. Preparation of the DNA

30 9

VI. Mapping Genes

31 0

VII. Overgo Strategy for Rapidly Doing Chromosomal Walks an d

Positional Cloning

317

VIII. Protocol for Overgo Probing of High-Density Filters

31 8

IX. General Flow of Information from the Radiation Hybrid Panel Maps ,

the Sanger Institute Sequencing Project, and Fingerprinting the BACs

32 1

X. Synteny Between Human, Zebrafish, and Fugu Genomes

325

XI. Proving a Candidate Gene Is Responsible for the Mutant Phenotype

326

XII. Morpholinos

327References

32 8

PART III Transgenesi s

18 . Lessons from Transgenic Zebrafish Expressing the Green Fluorescent Protein (GFP )

in the Myeloid Lineag e

Karl Hsu, A . Thomas Look, and John P. Kank i

I. Introduction

33 4

II. Myeloid Cells in Zebrafish

33 4

III. Expression of pu .1 in Myeloid Development

33 5

IV. Analysis of the Zebrafish pu .1 Locus

33 6

V. Germline Expression of Enhanced Green Fluorescent Protein (EGFP)

Under Control of the Zebrafish pu .1 Promoter

33 8

VI. Conclusions

345

References

346

19 . Sleeping Beauty Transposon for Efficient Gene Deliver y

Spencer Hermanson, Ann E . Davidson, Sridhar Sivasubbu, Darius Balciunas, an d

Stephen C . Ekke r

I. Introduction

34 9

II. Transgenesis Constructs

35 0

III. Microinjection of the Zebrafish Embryo

35 1

IV. Raising Injected Embryos

35 5

V. Identifying Transgenic Founders

35 6

VI. Visualizing Fluorescent Reporters

35 7

VII. Molecular Characterization of the Transposon Integration Site

35 8

References

36 1

20 . Transgene Manipulation in Zebrafish by Using Recombinases

Jie Dong and Gary W. Stuart

I. Introduction

36 3

II. Recombinase-Mediated Transgene Exchange, and Mobilization

36 7

III. Summary and Conclusion

37 5

References

376

21 . Highly Efficient Zebrafish Transgenesis Mediated by the Meganuclease I-Sce l

Clemens Grabher, Jean-Stephane Joly, and Joachim Wittbrodt

I. Introduction

38 1

II. Transgenesis by Meganucleases

38 6

References

39 8

22 . Cloning Zebrafish by Nuclear Transfer

Bensheng Ju, Haigen Huang, Ki-Young Lee, and Shuo Lin

I. Introduction

403

II. Recipes for Cell Culture and Nuclear Transfer

404

III. Cell Culture

404

IV. Nuclear Transfer

405

V. Summary of Nuclear Transfer

40 8

VI. Potential Applications of Zebrafish Cloning

409

References

410

PART IV Informatics and Comparative Genomics

23 . Data Mining the Zebrafish Genome

Lynn M. Schriml and Judy Sprague

I. Introduction

41 5

II. National Center for Biotechnology Information (NCBI) Tools ,

Resources, and Data Sets ,

41 6

III. Zebrafish Information Network (ZFIN)

42 8

IV. Tutorial

43 3

References

437

24 . The Zebrafish DVD Exchange Project: A Bioinformatics Initiative

Mark S . Cooper, Greg Sommers-Herivel, Cara T. Poage, Matthew B . McCarthy,

Bryan D . Crawford, and Carey Phillips

I. Introduction

440

II. Economic Aspects of Visual Bioinformatics

440

III. Building Infrastructures to Access Images

442

IV. Prior Experiences with Visual Data Dissemination (Mark Cooper)

443

V. Goals of the Zebrafish DVD Exchange Project

445

VI. Production of the Compact Disc (CD) Set : :"Zebrafish: The Livin g

Laboratory" (Greg Sommers-Herivel)

447

VII. Visual Bioinformatics and Databases

451

VIII. Internet2

45 2IX. Hyper Text Markup Language (HTML) and eXtensible Markup Languag e

XML (Bryan Crawford)

45 3X. Virtual Reality and Data Visualization Tools (Carey Phillips)

45 4XI. Summary

45 6References

45 7

25 . Comparative Genomics-An Application for Positional Cloning of th eweissherbst Mutan t

Anhua Song and Yi Zho u

I. Introduction

45 9II. Resources for Comparative Genomics Studies

460III. An Example of Using Comparative Genomic Resources in a Positiona l

Cloning Project

46 1References

47 3

26 . Comparative Genomics in Erythropoietic Gene Discovery : Synergisms Between th e

Antarctic Icefishes and the Zebrafish

H. William Detrich, III and Donald A . Yergea u

I. Introduction

47 6II. Representational Difference Analysis (RDA) Protocol

47 7III. Analysis of Unidentified RDA Contigs

48 5

IV. Isolation of Full-Length cDNAs Corresponding to RDA Products

49 3V. Model Hopping: Functional Analysis of the Zebrafish Ortholog of the

Novel Antarctic Fish Gene bloodthirsty

49 8VI. General Considerations

50 0References

50 1

27 . Spatial and Temporal Expression of the Zebrafish Genome by Large-Scale In Sit uHybridization Screenin g

Bernard Thisse, Vincent Heyer, Aline Lux, Violaine Alunni, Agnes Degrave, Iban Seiliez ,Johanne Kirchner, Jean-Paul Parkhill, and Christine Thiss e

I. Introduction and Goals

50 6II. Preparation of Antisense Digoxigenin (DIG)-Labeled RNA Probes

50 6III. Preparation of Embryos

50 8IV. Reagents and Buffers for In Situ Hybridization

50 9V. In Situ Hybridization Protocol

51 0VI. Double In Situ Protocol

514

VII. Recording Results

51 7

VIII. Concluding Remarks

51 7

References

51 9

28 . Design, Normalization, and Analysis of Spotted Microarray Dat a

F. B . Pichler, M. A . Black, L. C. Williams, and D . R . Lov e

I. Introduction

52 2II. Design of Microarray Experiments

52 2III. Array Preprocessing

53 0IV. Analysis of Microarray Experiments

53 5V. Discussion

54 0References

542

29. Comparative Genomics, cis-Regulatory Elements, and Gene Duplicatio n

Allan Force, Cooduvalli Shashikant, Peter Stadler, and Chris T. Amemiy a

I. Introduction

545

II. Comparative Genomics and Identification of cis-RegulatoryElements (CREs)

546III. Duplicated Genomes, Duplicated Genes

55 2References

55 6

PART V Infrastructure

30 . Zebrafish Facilities for Small and Large Laboratorie s

Bill Trevarro w

T . Introduction

566II. Zebrafish Biology

568

III. Fish Housing

569

IV. Quarantining

586V. Injections

587VI. Mutagenesis Room

587VII. Treating Outflow

588

VIII. Disposing of the Dead

588

IX. Disease Prevention

589

X. Cleaning

590XI. Human Safety

590References

591

31 . A Nursery that Improves Zebrafish Fry Surviva l

Peter Cattin and Phil Crosier

I. Introduction

59 3II. General Methods for Raising Embryos and Larvae

59 4III. A Nursery that Improves Fry Survival

59 4References

59 8

32 . Genetic Backgrounds, Standard Lines, and Husbandry of Zebrafis h

Bill Trevarrow and Barrie Robison

I. Introduction

599II. Nomenclature and Definitions

602III. Goals for Line Use

603IV. Breeding Strategies

60 4V. Cryopreservation

61 2VI. Monitoring and Response

61 3VII. Distribution

61 4VIII. Summary and Recommendations

61 4References

61 5

33 . Common Diseases of Laboratory Zebrafis h

Jennifer L . Matthews

I. Introduction

61 7II. Diagnostic Evaluation

61 8III. Common Diseases of Laboratory Zebrafish

61 8IV. Zoonosis

640References

641

34 . Zebrafish Sperm Cryopreservation

Stephane Berghmans, John P. Morris IV, John P. Kanki, and A . Thomas Loo k

I. Introduction : Benefits of Zebrafish Sperm Cryopreservation

64 5II. Critical Variables Affecting Sperm Cryopreservation

64 6III. Zebrafish Sperm Cryopreservation with N,N-Dimethylacetarnide

65 0IV. Future Directions

65 6References

65 8

Index

66 1

Volumes iii Series

679