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1、1 THE ZEBRAFISH BOOK A guide for the laboratory use of zebrafish Danio* (Brachydanio) rerio by Monte Westerfield, Institute of Neuroscience, University of Oregon *Older literature referred to zebrafish as Brachydanio rerio. Recent work (Meyer et al., Proc. Roy. Soc. Lond 252:231-236, 1993) and a con
2、sensus vote at the 1993 Zebrafish Meeting at the Cold Spring Harbor Laboratory suggest that Danio rerio should now be used. George StreisingerGeorge Streisinger This book is dedicated to the late George Streisinger who first began the study of zebrafish in Eugene and whose insight and gentle support
3、 got us all started. Adapted for the Web by ZFIN. Please address corrections and additions to: Monte Westerfield, Institute of Neuroscience, University of Oregon, Eugene, OR 97403. Copyright 1993 by Monte Westerfield. 4th Edition, 2000. Citing this information: Westerfield, M. (2000). The zebrafish
4、book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene. Hardcopies of the 4th edition of The Zebrafish Book can be obtained for a nominal fee from the Zebrafish International Resource Center, 5274 University of Oregon, Eugene, OR 97403 USA; fax: 541-3
5、46-6151. CONTENTS: (Click chapter title for contents) 1. GENERAL METHODS FOR ZEBRAFISH CARE 6 2. BREEDING 3. EMBRYONIC AND LARVAL CULTURE 4. MICROSCOPIC OBSERVATIONS 5. CELLULAR METHODS 6. DISSOCIATED CELL CULTURE 7. GENETIC METHODS 8. HISTOLOGICAL METHODS 9. MOLECULAR METHODS 10. RECIPES 2 11. REFE
6、RENCES THE ZEBRAFISH BOOK A guide for the laboratory use of zebrafish Danio (Brachydanio) rerio by Monte Westerfield, Institute of Neuroscience, University of Oregon 1. GENERAL METHODS FOR ZEBRAFISH CARE 2. BREEDING 3. EMBRYONIC AND LARVAL CULTURE 4. MICROSCOPIC OBSERVATIONS 5. CELLULAR METHODS 6. D
7、ISSOCIATED CELL CULTURE 7. GENETIC METHODS 8. HISTOLOGICAL METHODS 9. MOLECULAR METHODS 10. RECIPES 11. REFERENCES Chapter 1 - GENERAL METHODS FOR ZEBRAFISH CARE 1. Availability and simple care 6 2. Food 6 3. Water 7 4. Fish Diseases 8 o Velvet disease 9 o Mycobacteriosis 10 o Intestinal Capillarias
8、is 10 5. Quarantine Room Procedures 11 6. Cleaning Tanks 12 7. Shipping Fish 13 8. Keeping Track Of Stocks 22 9. Zebrafish Standard Protocol Form 25 Chapter 2 - BREEDING 1. Simple Method For Steady, Low-Level Embryo Production 25 2. Method For Maximal Embryo Production 26 3. Embryo Collection 26 4.
9、Simple Schedule For Breeding Zebrafish 27 5. Zebrafish Breeding Schedule For Maximal Embryo Production 27 6. Detailed Methods For Breeding Over Marbles 27 3 7. Pair-Wise Breeding Of Individual Or Natural Cross Fish 29 8. Embryo Production By In Vitro Fertilization 30 o Overview of in vitro fertiliza
10、tion 31 o General Procedures and Overview 32 o Squeezing Males 33 o Squeezing Females 34 o In Vitro Fertilization 35 Chapter 3 - EMBRYONIC AND LARVAL CULTURE 1. A Simple Method for Raising Babies 36 2. Raising Larvae in a Nursery 37 3. Simple, Quick Methods For Raising Paramecia 41 4. A Semi-Sterile
11、 Method For Raising Paramecia 41 5. Device for harvesting paramecia 42 6. Growing Paramecia Under Sterile Conditions 43 7. Paramecia Culture Medium 44 8. Purification of Paramecia 45 9. Microworms as a Food Source 46 10. A Simple Method for Raising Larvae 48 11. Developmental Staging Series 49 Chapt
12、er 4 - MICROSCOPIC OBSERVATIONS 1. Removing Embryos From Their Chorions 126 2. Viewing Chambers 126 3. Agar Mounting 126 4. Methyl Cellulose Mounting 127 Chapter 5 - CELLULAR METHODS 1. A Device to Hold Zebrafish Embryos During Microinjection 128 2. Blastomere Lineage Analysis 130 3. DiI Labeling 13
13、4 4. Labeling Single Cells With Lineage Tracers 135 5. Laser-ablation of Single Cells 138 6. Overview of Single Cell Transplantation 139 7. Detailed Procedure for Transplanting Single Cells 140 8. An Inexpensive and Easy Microinjection Embryo-Tray 143 Chapter 6 - DISSOCIATED CELL CULTURE 1. Preparin
14、g Embryos for Cell Culture 144 2. 0 Ca2+ - Mechanical Dissociation 144 3. Protease Dissociation 145 4. Dissociation of Embryos by Dissection 145 5. Using Coated Glass Coverslips for Cell Culture 145 6. Recipes: 146 4 o Embryo Extract o Growth Medium Chapter 7 - GENETIC METHODS 1. Conventions For Nam
15、ing Zebrafish Genes 146 2. Suggested Guidelines For Maintaining Mutant Stocks 148 3. Overview Of Methods For Parthenogenesis 150 4. Production Of Haploid Embryos 152 5. Production Of Homozygous Diploid Embryos 153 6. Heat shock 153 7. Early pressure 155 8. Delayed In Vitro Fertilization Using Coho S
16、almon Ovarian Fluid 157 9. Production Of Androgenetic Haploids and Diploids 160 10. Freezing Sperm 162 11. Thawing And Using Frozen Sperm For In Vitro Fertilization 163 12. Fin Amputations 164 13. Gamma Ray Mutagenesis 164 14. Chemical Mutagenesis 165 15. Testosterone Treatment To Produce Males 166
17、16. Genetic Strains 167 Chapter 8 - HISTOLOGICAL METHODS 1. Preparing subbed slides for sections 167 2. OCT Embedding for Cryostat Sectioning of Embryos or Larvae 168 3. Agar Embedding for Cryostat Sectioning of Embryos or Larvae 168 4. Gelatin Embedding for Vibratome Sectioning of Embryos or Larvae
18、 169 5. Staining Sections with Antibodies Using PAP 169 6. Staining Whole-Mount Embryos with PAP 169 7. Solutions for Antibody Staining Protocols 172 8. Whole-Mount Staining of Biotin-Dextran Injected Zebrafish Embryos 173 9. Photoconversion of Fluorescently Labeled Profiles for EM 174 10. Chromosom
19、e Spreads 175 11. Fixation by Freeze Substitution 177 12. Zebrafish Monoclonal Antibodies 178 Chapter 9 - MOLECULAR METHODS 1. Extraction Of Proteins From Zebrafish Embryos For SDS-Gel Analysis 183 2. Western Blots Of Zebrafish Embryos 184 3. Preparation Of Genomic Dna 187 4. Preparation Of Genomic
20、Dna For Southern Analysis 190 5. Rapid Isolation Of Rna From Zebrafish 190 6. Extraction And Purification Of Rna From Zebrafish Embryos 191 7. Preparation Of High Quality Rna From Zebrafish Embryos 191 8. Whole-Mount In Situ Hybridization 192 9. High Resolution Whole-Mount In Situ Hybridization 196
21、10. Two Color Whole-Mount Rna In Situ Hybridization 200 5 11. Sections Of Whole Mount In Situ Hybridization Preparations 205 12. Total Nucleic Acid Extraction Procedure For Zebrafish Embryos 206 13. A Simplified Ribonuclease Protection Assay For Zebrafish Embryos 206 Chapter 10 - RECIPES 1. ABC Solu
22、tion (Avidin-Biotin HRP Complex) 209 2. Agar/Sucrose 209 3. Atabrine Stock Solution 209 4. Blocking Solution for Western Blots 209 5. BSA/PBS/DMSO 209 6. BT Fix 209 7. Chorion Removal 209 8. DAB (Diaminobenzidine) 210 9. DAB Heavy Metal Stain 210 10. DAB Presoak Solution 210 11. DAB Solution 210 12.
23、 DNA Extraction Buffer 211 13. EDTA 211 14. Egg Water 211 15. Embryo Extract 211 16. Embryo Medium 211 17. Epon 211 18. Epon/Araldite 212 19. Finquel (MS222) 212 20. Fish Water 212 21. Fix Buffer 212 22. 1.25X Fix Buffer 212 23. Fixatives 212 24. Gelatin Embedding Medium 213 25. Genomic DNA Extracti
24、on Buffer 213 26. GHCl Buffer 213 27. Giemsa 213 28. Ginzburg Fish Ringers 213 29. GIT Buffer 213 30. Growth Medium 214 31. Hanks (Final) 214 32. Hanks (Full Strength) 214 33. Hanks Premix 214 34. Hanks Stock Solutions 214 35. Heat Shock (Diploid eggs) 215 36. In situ Hybridization Staining Buffer 2
25、15 37. Paramecia Seed Cultures 215 38. Paramecia for Baby Fish (Traditional Method) 216 39. Paramecia for Baby Fish (Streamlined Procedure) 216 40. Paramecia Medium Stock Solution I 216 41. Paramecia Medium Stock Solution II 216 42. Paramecia Storage Medium (Long Term) 217 43. PBS 217 44. 2X PBS 217
26、 45. PBS/BSA/DMSO 217 6 46. PCR Extraction Buffer 217 47. PMSF 217 48. PO4 buffer (0.1 M, pH 7.3) 218 49. Pronase 218 50. Protein Extraction Buffer 218 51. PTU 218 52. Ringers Solutions 218 53. Salt Stock 219 54. SDS Sample Buffer 219 55. Schnefelds Medium for Growing Paramecia 219 56. Sodium Bicarb
27、onate 219 57. Sperm Freezing Medium 219 58. Sperm Thawing Medium 220 59. Stock salts 220 60. Subbing Solution for Slides 220 61. TBS 220 62. TES buffer 220 63. Tricaine 220 64. TTBS 221 65. Western Blot color development buffer 221 66. Western Blot color development solution 221 67. Whole-Mount in s
28、itu Hybridization Solutions 221 12. CHAPTER 1 GENERAL METHODS FOR ZEBRAFISH CARE Availability and simple care Zebrafish are available at pet stores throughout the world. They can be most easily maintained in 10 gallon (45 liter) aquaria heated to 28.5C (above 31C and below 25C, zebrafish probably wo
29、nt breed and development will be abnormal) with 25 fish per tank. If you replace 1/3 of the water each day by siphoning up debris from the bottom of the tank, a separate tank filtering system will not be necessary. Otherwise use a filter and replace about half the water at least once a week. Tap wat
30、er, aged a day or more in an open (heated) tank to release chlorine, is adequate although more consistent conditions may be obtained by adding commercial sea salts to deionized or distilled water (60 mg of Instant Ocean per liter of water, for example). Adults should be fed 1-2 times per day with a
31、variety of food (see below). It is a good idea to clean the tank by siphoning after the second feeding. Food (Source: M. Westerfield) To keep fish in good breeding condition it is best to feed them a variety of foods. Feed manually ground dry or moist trout pellets (Ranger 1/4 inch brood food or Ore
32、gon wet pellets) as well as dry flake food like Tetra brand (available at most pet stores). Add enough food to each tank so that all the fish get some and all the food is eaten within 5 minutes. Feed adults at least twice a day although multiple light feedings allow the fish better opportunity to ut
33、ilize the food. The best possible food for breeding adults is live adult brine shrimp. Brine shrimp.i.Brine shrimp; eggs can be purchased at most pet shops. To make baby shrimp, add 10 ml of shrimp eggs to 2000 ml of salt water (or Instant.i.Instant Ocean; Ocean, Aquarium Systems Inc.). Aerate vigor
34、ously. After 48 hr at 28.5ree;C, 7 filter the shrimp through a cloth, wash with fresh water and dilute into dH2O at a ratio of 1 volume shrimp to 3 volumes water. Feed 1 pasteur pipette of diluted shrimp per 8 adult fish. Other possible foods are daphnia, Drosophila and Drosophila larvae. Beware of
35、tubifex worms which may carry diseases (also, see Raising Larvae in a Nursery). Water (Source: M. Westerfield) Several different types of water are used for procedures described in the following sections. These solutions have been adapted to balance ease and cost of production with the changing need
36、s of zebrafish during their life cycle. In general, adults can be maintained in tap water, conditioned by letting it set. This depends critically on the quality of the local water source and on the demands for embryo production. Poor water quality will adversely affect the health of the fish, their
37、susceptibility to disease, and their breeding potential. If there is any question about water quality, deionized or distilled water should be used to which a small amount of salts and minerals are added. If an adequate supply of deionized water is unavailable, a closed-system which recirculates the
38、water after purification can be used. Embryos and young larvae have stricter requirements and should be raised in egg water. Embryos removed from their chorions require additional calcium and should be maintained in embryo medium. System (or tank) water: water out of the facilitys water system. This
39、 water is dripped into clean tanks and is used for setting up pairwise crosses. Egg water: Used for in vitro fertilization and raising young embryos. Stock salts: 40 g “Instant Ocean“ Sea Salts added to 1 L distilled water Egg water = 1.5 ml stock salts added to 1 L distilled water = 60 g/ml final c
40、oncentration. Embryo medium: Dont confuse with “egg water“ above. Used in handling dechorionated embryos and storing young embryos in dishes. This is basically 10% Hanks with full strength calcium and magnesium. Embryo medium 1.0 ml Hanks Stock #1 0.1 ml Hanks Stock #2 1.0 ml Hanks Stock #4 95.9 ml
41、dd H2O 1.0 ml Hanks Stock #5 1.0 ml fresh Hanks Stock #6 Use about 10 drops 1 M NaOH to Ph 7.2 Hanks solutions: Hanks solutions can be made from stock solutions (kept refrigerated, they will last for several months). A premix of the salts can be stored in the refrigerator for several weeks. Sodium b
42、icarbonate does not store well, so it is made up fresh each time Hanks solution is made. Full Strength Hanks 0.137 M NaCl 5.4 mM KCl 0.25 mM Na2H PO4 0.44 mM KH2 PO4 1.3 mM CaCl2 8 1.0 mM Mg SO4 4.2 mM NaH CO3 Hanks Stock Solutions Stock #1 8.0 g NaCl 0.4 g KCl in 100 ml ddH2O Stock #2 0.358 g Na2HP
43、O4 Anhydrous 0.60 g KH2PO4 in 100 ml ddH2O Stock #4 0.72 g CaCl2 in 50 ml H2O Stock #5 1.23 g MgSO4-7H2O in 50 ml ddH2O Stock #6 0.35 g NaHCo3 10.0 mls dd H2Oz Hanks Premix - Combine the following in order: 10.0 ml Solution #1 1.0 ml Solution #2 1.0 ml Solution #4 86.0 ml ddH2O 1.0 ml Solution #5 St
44、ore Hanks Premix in the refrigerator along with the Hanks solutions. Final Hanks 9.9 ml Hanks Premix 0.1 ml fresh Stock #6 Fish Diseases (Source: C. Walker) 9 For any large colony of fish, precautions should be taken to prevent the spread of epidemic disease. The easiest strategy for combatting dise
45、ase is prevention by minimizing contact between fish and water in different tanks. Avoid mixing fish from different tanks as much as possible. Sterilize all equipment that comes in contact with the fish or tanks. For example, use fish nets, siphons, and cleaning sponges on only one tank at a time an
46、d autoclave them before using them in a different tank. Sterilize the water (i.e. with a flow-through ultra-violet sterilizing lamp) before adding it to the tanks. Remove sick fish from tanks as quickly as possible. Quarantine fish from pet stores before adding them to the colony (see Quarantine Roo
47、m Procedures, page 1). Wash hands and arms thoroughly if they come into contact with tank water. The two most common diseases that affect zebrafish are velvet disease and fish tuberculosis (mycobacteriosis). Velvet disease Zebrafish are highly susceptible to velvet disease, Oodinium pillularis, a pa
48、rasitic dinoflagellate algae. This oval-shaped parasite attaches to the fish near the fins, especially the dorsal fin, and around the gills. You can see it under a dissecting microscope. When the parasite is mature, it drops off the fish and multiplies 60 times on the bottom of the tank. These new p
49、arasites then reinfect the fish. Fish with velvet disease have the characteristic behavior of rubbing their sides and flipping around in the corners of the tank. As the disease progresses, fish become lethargic, the fins (particularly the dorsal fin) are held close to the body, and the fish stop producing eggs. Most fish from pet stores or dealers carry velvet disease. Symptoms of velvet disease: rubbing behavior lethargy fins held close to the body parasites near