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Cell/Tissue Transplantation in Zebrafish 176. Agar (e.g., DIFCO [Frankiln Lakes, NJ] BACTOAGAR): dissolved in distilledwater or the desired Ringer’s solution.7. Antibiotics: penicillin and streptomycin solution (10000 U/mL penicillin and10,000 µg/mL streptomycin, Gibco BRL [Rockville, MD] 15140-122) are addedto all media used for operations at a final concentration of 1% to 2%.8. Methyl cellulose (e.g., 3500–5600 cps, Sigma [St. Louis, MO] M-0387).9. Lipofectamine (Gibco BRL 18324-012).10. Rhodamin-dextran (10,000 MW, e.g., Molecular Probes, [Eugene, OR] D-1816).11. Biotin-dextran (10,000 MW, lysine fixable; e.g., Molecular Probes, D-1956).12. Albumen, prepared from egg white: Addition of egg albumen to Ringer’s solutionsometimes increases the survival rate of embryos which have been manipulated,especially when the embryos have sustained some damage to the yolkmembrane by the removal of the yolk or fusion of two embryos (8). In addition tonutritive components, the albumen contains lysozyme, a bacteriostatic agent. Forthis reason, egg albumen is often used in embryo cultures to prevent the growthof microorganisms as well as for nutrition.13. Embryo transfer pipet: Pasteur pipets and rubber teats.14. 35-mm, 60-mm, and 100-mm plastic culture dishes with lids.15. Agar-coated dishes for dechorinated embryos: Pour an appropriate amount of hot1% agar in the desired Ringer’s solution into culture dishes and wait until it iscompletely solidified. Fill the dishes about three-quarters full with the desiredRinger’s solution. Agar-coated dishes help to prevent the embryos from stickingto the dish.16. Micromanipulator: A simple manual micromanipulator works well for cell transplantation(e.g., Narishige, MM-3).17. Watchmaker’s forceps.18. Sharpened glass needle: The end of a Pasteur pipet is pulled to a fine tip on asmall gas burner or spirit lamp.19. Blunt glass needle: Burn the tip of sharp glass needle for a while.20. Tungsten needle: sharpened from a fine tungsten filament (0.2 mm in diameter,e.g., Nilaco Corp., Tokyo). To sharpen, mount into a Pasteur pipet or needleholder, then insert repeatedly in the side of a very hot flame; further sharpen byrepeatedly soaking the tip of the filament in melted sodium nitrite. For melting,heat the crystal in a quartz melting pot with a gas burner. Do not use ceramicpots, which cannot withstand the heat of melting sodium nitrite. This process isvery dangerous and great care should be taken.21. Mold for making holes in agar-coated dishes (Fig. 1A): agar-coated dishes containingmultiple holes are required for holding embryo/yolk cell combinations toensure complete adhesion between the donor and host tissues. The holes in theagar should just fit the recombinants. The best diameter for the hole is approximately1.2 mm. To make these dishes, we use a silicone rubber mold. The siliconemold is made by pouring liquid silicone mixed with a hardener onto astainless plate containing holes ( = 1.2 mm), in which one end of the hole hasbeen sealed with tape (Fig. 1B).
18 Mizuno, Shinya, and TakedaFig. 1. Transplantation of the yolk cell. (A) A silicone rubber mold for agarholes. Scale bar = 10 mm. (B) A stainless steel plate used for production of thesilicone mold shown in A. The diameter of the hole is 1.2 mm. (C) Schematicrepresentation of the experiment. (D–G) The process of adhesion between the donoryolk cell (upper) and the host embryo (lower) which are kept in an agar hole.Scale bar = 100 µm.22. Hooked glass needle (Fig. 2A) used for removal of the yolk mass: Glass capillariesare pulled to fine tips on an electrode puller. The tips are then fire polishedwith a microforge. To make a hooked shape, heat the center of the pulled capillarywith a microforge.
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Page 3 and 4: 2 Greentally active genes. Importan
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WISH of Xenopus and Zebrafish Embry
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70 Bertwistleparative techniques. F
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72 Bertwistle3. Methods3.1. In Vitr
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76 Bertwistlegenes along the dorsov
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78 MacdonaldFig. 1. Immunohistochem
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80 Macdonaldlarger experiments, use
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82 Macdonaldblocked. This requires
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84 Macdonald9. The detection step i
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86 Macdonaldattached to the seconda
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88 Macdonaldparticular, I would lik
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92 Robinson and Guille6. BM Purple
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Synthetic mRNA for Microinjection 9
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112 Guilleantibodies (16), and anti
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118 Guille4. The next morning, the
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120 Guille10. Incubate the oocytes
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122 Guilleencoding a protein (4F2hc
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Microinjection into Zebrafish Embry
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Expression from DNA Injected into X
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158 JooreFig. 2. (A) A plastic mold
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160 Joorements). Be sure to adjust
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162 Joorethe yolk cell. Second, for
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164 Joore2. Microinjection in zebra
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166 Joore5. Kroll, K. L. and Amaya,
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168 Fu, Kan, and Evansconstruction
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170 Fu, Kan, and Evans2. MaterialsA
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172 Fu, Kan, and EvansITRs, we use
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Band-Shift Analysis of Oocyte and E
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DNA Footprinting Using Embryonic Ex
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