To obtain X. laevis embryos, female frogs were induced to ovulate by injecting 500 to 700 U of human chorionic gonadotropin subcutaneously, directly above the dorsal lymph sacs. After injection, females were maintained in the dark at 18°C overnight. After females began ovulating the following morning, one male was euthanized by placing it in 0.1% tricaine solution buffered with sodium bicarbonate for 30 min. Both testes were dissected and placed into a 35-mm dish containing 1.5 ml of 1× Mark’s modified ringer (MMR) solution [0.1 M NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, and 5 mM Hepes (pH 7.8)]. Eggs were gently squeezed from females into 10-cm plates. Sperm were released to fertilize the eggs by mincing a small portion of the testes in a few drops of 1× MMR placed beside the eggs followed by gentle mixing of eggs and sperm together. After 10 min at room temperature (RT), the fertilized eggs were flooded with 0.1× MMR solution (pH 7.4). Embryos were dejellied using 2% l-cysteine solution (pH 7.8) for 2 min. Embryos were washed thrice with 0.1× MMR solution and placed in 0.1× MMR. At the desired developmental stage, embryos were injected with a Nanoject II Auto-Nanoliter Injector (Drummond Scientific Inc.). Injected embryos were maintained in 4% Ficoll/0.1% MMR solution at 18°C until they reached stage 9 and then transferred to 0.1× MMR buffer.

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