Part 4: Fertilization and Development

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Protocol submitted by Janet Heasman [1]

Fertilize embryos

Three hours after the oocytes are transferred to the host frog, the frog is squeezed to lay eggs at 20-30 minute intervals and fertilized with a sperm suspension, as per a normal in vitro fertilization (Holwill et al., 1987). The percentage of experimental oocytes that are laid by the female varies from one experiment to the next, but as long as the female does not stop laying eggs most of the dyed oocytes can be recovered. Of these only a portion of them will fertilize, and a 20-40% rate of fertilization is usual for transferred and uninjected oocytes. Usually a female will give 3-4 good fertilizations per experiment.

Dejelly embryos

Embryos are dejellied in 2% cysteine pH 7.8 in 0.1X MMR (Appendix A) after the first cell cycle. Briefly, they are gently swirled until the embryos lie close together instead of being held apart by the jelly coat. Then embryos are washed three times in a large excess volume of 0.1X MMR. Both host and transferred embryos are then allowed to develop in 0.1XMMR.

As it is easier to distinguish the 5 colors from each other early in development rather than later, the embryos are subdivided into separate dishes at this time and are monitored carefully through development to observe differences between the control and experimental embryos. In addition, embryos may be fixed and frozen for further analysis at later stages.

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