Fish Raising and Embryo Manipulation

1. Tanks and water (see Note 2): Systems for rearing and maintaining zebrafish range from commercially available modular systems to small-scale, self-built facilities (8,12). What is of most importance, however, is water quality. To manage a diploid screen efficiently, fish must lay in a high percentage of pair matings. This seems to relate, at least in part, to the water quality. Tap water quality can be greatly improved by the use of filtration systems. Charcoal filters seem to be neccesary with sand filters proving a useful second-step filtration, although neither of these can buffer against regional differences in water quality, such as heavy metal content. Water quality should be assayed before facility designs are too advanced to accommodate any specialized needs. One valuable addition to consider is a UV sterilizer, which can greatly aid in keeping stocks disease-free.

It is also important that pH is kept neutral or slightly high, since zebrafish are sensitive to an acidic environment, but seem to tolerate mild alkaline water.

There are many varieties of tank designs. In a diploid screen where many pair matings are performed, it is vital to have an efficient system to set up breeding pairs to collect embryos. We use small plastic boxes with wire mesh replacing the bottom of one box and stacked inside another. Eggs fall through the mesh, and parents are restrained from eating them. Pairs or individual females are kept separate in small plastic boxes. Larger 4-L mouse cages are used for rearing of juvenile fish, and adult fish are kept in larger permanent glass tanks of the facility. A serial system of glass tanks with a commom water-flowthrough is the most efficent design for facilities with space constraints (see Note 7).

2. Food: There are also many diets that can be used. Adults do well on most commercial flake diets, and we use Tetramin flakes finely ground with a mortar and pestle to a powder. Juveniles from about 3 wk of age are fed hatched live brine shrimp, and these can be fed to the adults also. Care must be taken to remove all unhatched brine shrimp, since these are not easily digested. Fry from a week old are fed alternatively filtered paramecium cultures and a commercial protein diet of liquifry (Tetramin) and Tetramin fry food mixed. Fish of all stages are fed twice a day. Sexual maturity can be achieved much more quickly if the number of feedings and food density is kept high to juveniles. However, adult fish fed in this manner may have a shorter life cycle and stop breeding more quickly. This can be a major problem at the end of a screen lasting a year or so, since tanks of females identified as carrying mutations may have stopped breeding before they can be out-crossed or sperm collected for freezing, in the case of the males.

3. Hank's solution: Full-strength Hank's solution is 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3. Bicarbonate is made fresh.

4. Embryo medium: This is 10% (v/v) Hank's with magnesium and calcium at full strength.

5. Methylene blue: A weak solution of methylene blue acts as an antifungal agent. A stock solution is made by diluting methylthione chloride powder (Sigma) to 2% (w/v) in dH2O. One or 2 drops are added/400-500 mL just until water turns pale blue. Higher concentrations result in uptake of the blue dye by developing embryos.

6. 1-Phenyl-2-thiourea (PTU): An active solution of PTU is made by dissolving the powder to 0.003% (w/v) in 10% Hank's. Fish must be raised in this solution from fertilization to inhibit pigment synthesis. PTU is also neurotoxic, and solutions and powder should be handled with care. Fish raised in this solution should be separated from the rest of the fish to avoid the possibility of PTU being placed into the system.

7. Tricaine: This is the most readily used fish anesthetic and, if applied correctly, seems to have no adverse side effects. A stock solution of tricaine is made by dissolving 3-amino benzoic acidethylester (Sigma) powder to 0.4% (w/v) in 20 mM Tris (pH 9.0) and adjusting the pH to 7.0. This solution is stored at -20°C

and diluted to approx 0.015-0.020% (v/v) in water to provide a working solution. The stock solution loses its efficacy over time and should be made up monthly. Tricaine is also a mild carcinogen. Although it is impossible to avoid contact with the solution in manipulations involving its use, care should be taken to avoid overexposure.

8. Methyl cellulose: A 3% (w/v) solution of methyl cellulose can be used to mount and photograph live anesthetized embryos. Powder is dissolved in water via gentle heating to a viscous solution. The solution is refrigerated to remove air bubbles and then brought to room temperature before use. Fish can simply be removed from the methyl cellulose by sequential rinses in water.

9. UV source: Any source of shortwave UV light can induce haploidization in zebrafish embryos as long as this effect is calibrated. The source used in our laboratory is Ultraviolet Products Incorporated UVL-56.

10. Fish Ringers. Fish Ringers is 0.11 M NaCl, 0.0034 M KCl, CaCl2(H2O)2, and 0.0024 M NaHCO3. Bicarbonate is added last and immediately before use.

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