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1、 THE ZEBRAFISH BOOK A Guide for the Laboratory Use of Zebrafish Danio (Brachydanio) rerio by Monte Westerfield, Institute of Neuroscience, University of Oregon 247 CHAPTER 1 1 GENERAL METHODS FOR ZEBRAFISH CARE 1 Availability and simple care 1 Food 1 Water 1 Fish Diseases 3

2、 Quarantine Room Procedures 6 Cleaning Tanks 7 Shipping Fish 8 Keeping Track of Stocks 15 Chapter 2 - Breeding 20 Simple Method For Steady, Low- Level Embryo Production 20 Method For Maximal Embryo Production 20 Embryo Collection 21 Simple Schedule For Breeding Zebrafish 21 A Zebrafish Bre

3、eding Schedule For Maximal Embryo Production 22 Detailed Methods for Breeding Over Marbles 22 Pair-Wise Breeding of Individual or Natural Cross Fish 24 Embryo Production By In Vitro Fertilization 26 CHAPTER 3 - EMBRYONIC AND LARVAL CULTURE 33 A Simple Method for Raising Babies 33 Raising Larva

4、e in a Nursery 33 Simple, Quick Methods For Raising Paramecia 38 A Semi-Sterile Method For Raising Paramecia 39 Growing Paramecia Under Sterile Conditions 41 Paramecia Culture Medium 42 Purification of Paramecia 43 Microworms as a Food Source 45 A Simple Method for Raising Larvae 46 CHAPTER

5、4 - MICROSCOPIC OBSERVATIONS 136 REMOVING EMBRYOS FROM THEIR CHORIONS 136 VIEWING CHAMBERS 136 AGAR MOUNTING 137 METHYL CELLULOSE MOUNTING 138 CHAPTER 5 - CELLULAR METHODS 140 A Device to Hold Zebrafish Embryos During Microinjection 140 Blastomere Lineage Analysis 142 DiI Labeling 147 Label

6、ing Single Cells With Lineage Tracers 148 Laser-ablation of Single Cells 151 Overview of Single Cell Transplantation 152 Detailed Procedure for Transplanting Single Cells 154 An Inexpensive and Easy Microinjection Embryo-Tray 157 CHAPTER 6 - DISSOCIATED CELL CULTURE 159 Preparing Embryos for C

7、ell Culture 159 0 Ca2+ - Mechanical Dissociation 159 Protease Dissociation 159 Dissociation of Embryos by Dissection 160 Using Coated Glass Coverslips for Cell Culture 160 Recipes: 160 CHAPTER 7 - GENETIC METHODS 162 Conventions for Naming Zebrafish Genes 162 Suggested Guidelines for Maintai

8、ning Mutant stocks 164 Overview of Methods for Parthenogenesis 166 Production of Haploid Embryos 169 Production of Homozygous diploid embryos 170 DELAYED IN VITRO FERTILIZATION USING COHO SALMON OVARIAN FLUID 175 PRODUCTION OF ANDROGENETIC HAPLOIDS AND DIPLOIDS 178 Freezing Sperm 182 Thawing

9、and Using Frozen Sperm for In Vitro Fertilization 183 Fin Amputations 184 Gamma Ray Mutagenesis 184 Chemical Mutagenesis 185 Testosterone Treatment to Produce Males 187 ZEBRAFISH STRAINS 188 CHAPTER 8 - HISTOLOGICAL METHODS 189 Preparing subbed slides for sections 189 OCT Embedding for Cryos

10、tat Sectioning of Embryos or Larvae 189 Agar Embedding for Cryostat Sectioning of Embryos or Larvae 190 Gelatin Embedding for Vibratome Sectioning of Embryos or Larvae 191 Staining Sections with Antibodies Using PAP 192 Staining Whole-Mount Embryos with PAP 193 Solutions for Antibody Staining P

11、rotocols 195 Whole-Mount Staining of Biotin-Dextran Injected Zebrafish Embryos 197 Photoconversion of Fluorescently Labeled Profiles for EM 198 Chromosome Spreads 200 Gentle Fixation by Freeze Substitution 201 The following is a list of available antibodies at ZIRC: 203 Chapter 9 - Molecular M

12、ethods 209 Extraction of Proteins from Zebrafish Embryos for SDS-Gel Analysis 209 Western Blots of Zebrafish Embryos 209 Preparation of Genomic DNA 213 Preparation of Genomic DNA for Southern Analysis 215 Rapid Isolation of RNA from Zebrafish 216 Extraction and Purification of RNA from Zebrafi

13、sh Embryos 216 Preparation of High Quality RNA from Zebrafish Embryos 217 Whole-Mount in situ Hybridization 218 High Resolution whole-mount in situ hybridization 222 TWO COLOR WHOLE-MOUNT RNA IN SITU HYBRIDIZATION 227 Sections of Whole Mount in situ Hybridization Preparations 233 Total Nucleic

14、 Acid Extraction Procedure for Zebrafish Embryos 234 A Simplified Ribonuclease Protection Assay for Embryos 235 CHAPTER 10 - RECIPES 237 CHAPTER 1 GENERAL METHODS FOR ZEBRAFISH CARE Availability and simple care Zebrafish are available at pet stores throughout the world. They can be most easi

15、ly maintained in 10 gallon (45 liter) aquaria heated to 28.5C (above 31C and below 25C, zebrafish probably won't 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 syste

16、m will not be necessary. Otherwise use a filter and replace about half the water at least once a week. Tap water, 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

17、water (60 mg of Instant Ocean per liter of water, for example). Adults should be fed 1-2 times per day with a 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 t

18、o feed them a variety of foods. Feed manually ground dry or moist trout pellets (Ranger 1/4 inch brood food or Oregon 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

19、minutes. Feed adults at least twice a day although multiple light feedings allow the fish better opportunity to utilize 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 m

20、l of shrimp eggs to 2000 ml of salt water (or Instant.i.Instant Ocean; Ocean, Aquarium Systems Inc.). Aerate vigorously. After 48 hr at 28.5°ree;C, 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 o

21、f diluted shrimp per 8 adult fish. Other possible foods are daphnia, Drosophila and Drosophila larvae. Beware of 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 t

22、he following sections. These solutions have been adapted to balance ease and cost of production with the changing needs 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

23、source and on the demands for embryo production. Poor water quality will adversely affect the health of the fish, their 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

24、and minerals are added. If an adequate supply of deionized water is unavailable, a closed-system which recirculates the 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 additiona

25、l calcium and should be maintained in embryo medium. System (or tank) water: water out of the facility's water system. This 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 "In

26、stant Ocean" Sea Salts added to 1 L distilled water Egg water = 1.5 ml stock salts added to 1 L distilled water = 60 µg/ml final concentration. Embryo medium: Don't confuse with "egg water" above. Used in handling dechorionated embryos and storing young embryos in dishes. This is basically 10% H

27、ank's with full strength calcium and magnesium. Embryo medium 1.0 ml Hank's Stock #1 0.1 ml Hank's Stock #2 1.0 ml Hank's Stock #4 95.9 ml dd H2O 1.0 ml Hank's Stock #5 1.0 ml fresh Hank's Stock #6 Use about 10 drops 1 M NaOH to Ph 7.2 Hank's solutions: Hank's solutions can be ma

28、de 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 bicarbonate does not store well, so it is made up fresh each time Hank's solution is made. Full Strength Hank's 0.137 M NaCl 5.4 m

29、M KCl 0.25 mM Na2H PO4 0.44 mM KH2 PO4 1.3 mM CaCl2 1.0 mM Mg SO4 4.2 mM NaH CO3 Hank's Stock Solutions Stock #1 8.0 g NaCl 0.4 g KCl in 100 ml ddH2O Stock #2 0.358 g Na2HPO4 Anhydrous 0.60 g KH2PO4 in 100 ml ddH2O Stock #4 0.72 g CaCl2 in 50 ml H2O Stock #5 1

30、.23 g MgSO4-7H2O in 50 ml ddH2O Stock #6 0.35 g NaHCo3 10.0 mls dd H2Oz Hank's 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 Store Hank's Premix in the refrigerator along with the Hank's solut

31、ions. Final Hank's 9.9 ml Hank's Premix 0.1 ml fresh Stock #6 Fish Diseases (Source: C. Walker) For any large colony of fish, precautions should be taken to prevent the spread of epidemic disease. The easiest strategy for combatting disease is prevention by minimizing contact between fish

32、 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 and autoclave them before using them in a different ta

33、nk. 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 Room Procedures, page 1). Wash hands and arms thoroughl

34、y 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 parasitic dinoflagellate algae. This oval-shaped pa

35、rasite 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 parasites then reinfect the fish. Fish with velvet

36、 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

37、 carry velvet disease. Symptoms of velvet disease: · rubbing behavior · lethargy · fins held close to the body · parasites near fins and gills Although velvet disease is extremely contagious, it can be cured with minimal damage to the fish using a 3-day treatment with Atabrine (Quinacrine

38、 hydrochloride). Treatment for velvet disease: · Day 1 · Turn off incoming water. · Slowly drip 2 liters of sea salts into an infected 10-gallon tank. · Add 3.3 ml of the Atabrine stock solution. · Day 2 · Add 3.3 ml Atabrine stock. · Day 3 · Add another 3.3 ml Atabrine stoc

39、k for a total of 9.9 ml. · At the end of the 3-day period, clean the bottom of the tank thoroughly and slowly dilute out the salt and the Atabrine with fresh water. · Continue cleaning the bottom of the tank daily for several days. · Solutions: · Atabrine Stock:10 mg/ml dH2O. Store in lig

40、ht tight bottle. · Salt Stock: 20 tablespoons (280 g) Instant Ocean Sea Salts (Aquarium Systems, Inc.) dissolved in 2 liters distilled water Mycobacteriosis Fish tuberculosis or mycobacteriosis is also a common disease. The agent, Mycobacterium marinum, is a rod-shaped, gram positive, non-motil

41、e, non-spore-forming bacterium1. Fish are infected by ingesting the bacteria in unpasteurized food or through abrasions in the skin. Snails often harbor the bacteria. Symptoms of Mycobacteriosis: o listlessness; the fish may isolate itself from other fish and refuse to eat o emaciation; hence,

42、 the name "skinny" o skin ulcers o spinal curvature o pigment change o grey-white nodules in internal organs Definitive diagnosis should be made by a pathology lab. There is no known effective antibiotic to treat mycobacteriosis. Some level of disease control can be obtained by eliminating

43、 sick fish, by sterilizing tanks routinely and all equipment that comes in contact with the fish or the tank water, and by reducing stress caused by moving fish between tanks or by changes in temperature, water flow, or feeding regimen. A word of caution regarding fish tuberculosis: Some cases of

44、transfer of fish tuberculosis to humans has been documented. Precautions should be taken by wearing disposable gloves and washing hands with betadine (providone iodine) when handling sick fish. Persons with immuno-deficiency problems should not handle fish with TB. Other diseases that occur less f

45、requently include raised scales, tumorous eyes, and hemorrhages. In these instances, remove the diseased fish as soon as they are seen and clean the tank. Reference: Post, G. (1987) Textbook of Fish Health, T.F.H. Publications, Neptune City, NJ, pp. 228. Intestinal Capillariasis (Source: M. Pac

46、k, J. Belak, C. Boggs, M. Fishman and W. Driever) Capillarids are thin and transparent worms that can reach one centimeter length. The eggs have a characteristic oval shaped appearance with a plug-like structure at either end and are visible in the adult worm, and the gut or feces of infected fish

47、. Photographs and a good description of Capillarids at different developmental stages can be found in the Handbook of Fish Diseases, Dieter Untergasser, Editor (TFH publications, 1989, p. 104-105). Worms in the intestinal bulb of adult zebrafish are motile and can be easily seen when the dissected g

48、ut, in egg water (0.03% Instant Ocean), is viewed with transmitted light using a high power (50X) dissecting microscope. Outside the fish gut Capillarids are no longer motile. A combination of two drugs, Trichlorfon and Mebendazole ("Fluke-tabs"; Aquarium Products, 180-L Penrod Court, Glen Burnie,

49、 M.D. 21061) has been reported to be extremely efficacious for removing monogenetic trematodes from fresh water fish. Trichlorfon is an insecticide with anti-cholinergic activity that is considered toxic to humans. Mebendazole, a common anti-helminthic used to treat human intestinal infections, inhi

50、bits glucose uptake and is cidal for adult helminths and embryos. Use the dosage recommended by the supplier; one tablet per 38 liters once trials to assess toxicity are completed. Add the drugs as a slurry (100 tablets per one liter water-let stir 10 minutes) because they are poorly soluble in wa

51、ter. Repeat the treatment after 24 and 48 hours with a 10% water change every day thereby increasing the effective concentration. Remove carbon filters during treatment. Wear plastic gloves to avoid contact with water. Reinstall carbon filters at 72 hours to remove the drugs from the system. Repeat

52、the treatment protocol at 10 days because the cidal effect of these drugs on freshly fertilized eggs or dauerlarvae is not reported. Continue testing fish at regular intervals and retreat if necessary. References: Goven, B.A. and Amend D.F. (1982) Mebendazole/trichlorfon combinations: A new an

53、ti-helminthic for removing monogenetic trematodes from fish. J. Fish Biol. 20:373-378. Quarantine Room Procedures (Source: S. Russell) Fish brought in from the outside world (i.e. not born in the facility) must be isolated in a quarantine room to reduce the risk of contaminating the existing st

54、ocks with infectious diseases. The following procedures are designed to prevent the accidental spread of diseases from the quarantine area. 1. Only pre-authorized people are allowed in the quarantine room. 2. All incoming fish are examined and, if necessary, treated for velvet disease. 3. All

55、new fish are to be checked daily for two weeks for signs of illness. Any potential sickies are removed. 4. Dead fish.i.Dead fish; are transported in a plastic bag and flushed down the sink in the quarantine area with lots of water. 5. No live adult fish are to be moved from the quarantine room u

56、nless they leave the building entirely. 6. Embryos obtained in the quarantine room (after the initial 2 week period) must be treated in bleach before leaving the room. To bleach eggs, prepare two beakers of bleach solution, containing 0.1 ml of 5% sodium hypochlorite in 170 ml of system water. Mix

57、 thoroughly. Place the eggs in the first beaker, and allow them to stand for 5 min. Pour off the bleach solution, and rinse the eggs with system water. Allow the eggs to stand in system water for 5 min. Place the eggs in the second beaker of bleach solution for 5 min. Rinse the eggs with system wate

58、r. Place the eggs into a small disposable petri dish. Eggs that have been properly bleached can be removed safely from the quarantine room. 7. No equipment can leave the quarantine area without being bleached or autoclaved. Equipment is transported, cleaned and stored in containers that are kept s

59、eparate from the central facility containers and are labeled "Q-room". No equipment is to be moved in or out of the Q-room without prior approval. 8. Always wash hands (and arms) carefully in the local sink using antiseptic soap after working in the quarantine room. 9. When working in the quaran

60、tine area, assume that everything (including yourself) is contaminated. Think about what you're doing! Cleaning Tanks (Source: T. Montgomery) Siphon (cleaning by "vacuuming") the bottoms of all tanks at least once per week. Use siphon tips with a length that corresponds to the size of the tank:

61、 tips for 5-gallon tanks are 12" long; the 10-gallon tips are 18" long; and the 30-gallon tips are 26" long. Each tip is made of 0.5" I.D. x 1/16" Wall Plexiglas tubing (Port Plastics, Portland, Oregon). Cover one end with a one inch tip of 3/8" I.D. x 3/32" Wall Amper Latex tubing at one end (VWR S

62、cientific Co.). (See Embryo Collection, for a diagram.) The siphon tips, which are attached to the hoses, are the only parts of this apparatus that can be immersed in the tank. Use each tip in only one tank and then sterilize it by soaking in a bleach solution (solution is 20 parts water to 1 part b

63、leach) followed by a rinse in a pipette rinser. Scrubbing (removal of algae from sides, front, and back of tank) is done on an "as needed" basis. (If you can't see into the tank, it's past time to scrub.) Make scrubber handles from 0.5" x 1.5" Plexiglas Plastic (Port Plastics, Portland, Oregon). T

64、he heads are made by sewing Scotch Brite Pads (United Grocers, Eugene, Oregon) with 20 lb test monofilament fishing line so they slip tightly over the scrubber handles. Use each handle and head assembly in one tank only and then sterilize by bleaching as described above for the siphon. The scrubber.

65、i.Scrubber; heads can be autoclaved. Use two lengths of scrubber handles. The size for 5 and 10 gallon tanks is 0.5" x 1.5" x 16" and for the 30 gallon tanks use 0.5" x 1.5" x 24" handles. Heads are the same for both size handles. Tanks that are emptied of fish need to be cleaned and sterilized be

66、fore another batch of fish can be introduced. Drain the tank and remove it from the rack. Remove and discard all tape, air pipettes, and siphon tube netting. All other parts (lid, back, and water fittings) are kept with that particular tank. Clean all parts with brushes and a scrub pad. Clean the tank thoroughly with a scrub pad, taking care not to damage the silicon water seals on the inside (algae should be left if very gentle rubbing will not remove it). Once the tank and parts are clean they