Cryopreservation of embryos

Cryopreservation of early embryonic stages (reprinted from Schenkel, Transgene Tiere 2. edition 2006, ISBN-10 3-540-28267-X, with friendly permit of Springer-Verlag, Heidelberg)
© dkfz.de

Embryos are prepared from superovulated female donors, loaded on a straw with medium and glycerol (serving as cryoprotectant) and will be frozen using a regulated freezer to -32°C. Afterwards the straws are plunged into LN2 and can be stored unlimited. Following revitalisation the embryos must be transferred into a pseudopregnant foster mother.
Depending on the genotype of the transgene a minimum of 200 to 500 embryos are needed for a sufficient cryopreservation, this means an average of 20 to 50 vaginal plug positive donors. In the individual case the number of donors required might be much higher. The gain of embryos depends on several factors as

  • facility/environment
  • genetic background
  • age of male
  • age of female
  • superovulation
  • possible phenotype
  • pheromonic effects
  • frequency of breeding
  • health condition
  • season/extreme weather conditions

Cryopreseration of 2- to 8-cell embryos has been qualified as “Gold Standard”. The examination of the quality of the frozen samples is necessary, but only possible with the loss of the corresponding sample. This might be a disadvantage in case of bad breeding lines. In case of not satisfying revitalisation ratios additional embryos are to be cryopreserved. Re-genotyping makes no sense if non-transgenic females serve as embryo donors.
Revitalisation: An average of 80% revitalisation is received in our hands. Depending on the embryo-transfer required in parallel a rederivation of the corresponding line is maintained. This technique is in the mouse model available for most of all genetic backgrounds.

to top