Valuable transgenic lines can be frozen and preserved indefinitely by cryopreservation. Cryopreservation protects the transgene line from environmental catastrophes, but is also an economical and labor-saving method of preserving lines for future detailed analysis. Mouse blastocysts can be generated by culturing fertilized one-celled embryos in M16 medium (12,22). Rat eggs do not survive very well in culture, so rat blastocysts are preferably obtained directly from the animal at 5 d post-hCG.

There are two general methods for cryopreservation of mouse embryos (23, 24). One is an equilibrium method (slow cooling), which uses a programmable cooling machine; embryos are exposed to moderate cryoprotectant concentrations and are cooled slowly at 0.3-2°C/min. Embryos are dehydrated during this slow-cooling process.

The second is a fast-cooling method that requires only a -70°C freezer: embryos are exposed to high molar concentrations of cryoprotectants and cooled rapidly. Exposure time to cryoprotectants is reduced and vitrification occurs soon after the cooling begins. This method is more sensitive to minor variations in protocol, especially time.

Taking into account the survival rate of the embryos as they undergo the different manipulations (e.g., from 100 frozen embryos, 88 are recovered from straws of which 77 survive freeze-thawing, and 7-8 pups are born after oviduct transfers of which 4 are transgenic), at least 400 embryos must be frozen in two different sessions to be sure of obtaining 15-16 transgenic animals.

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