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Embryo cryopreservation

Vivarium and Transgenics Core

Overview

Investigators interested in the mouse embryo cryopreservation service should have two to four fertile males, two to six months of age. The service begins once VARI receives a completed request form via iLab and the male mice. The males are housed individually for one week before the service begins. Strain-specific wild type females or females provided by the investigator will be used. The females are superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to maximize the number of harvested embryos. In vitro fertilization (IVF) is performed using the frozen sperm and the designated females.  If the mice are homozygous, Core personnel will freeze 250–300 embryos. If the mice are heterozygous, Core personnel will freeze 350–400 embryos. 2-cell stage embryos will be cryopreserved.

Cryopreservation method

IVF is performed with strain specific females. Healthy 2-cell stage embryos are collected and cryopreserved in straws (25–30 embryos per straw) using 1-2 Propaniedol, propylene glycol, PROH as the cryoprotectant and sucrose. Cryopreservation is performed using a controlled rate alcohol bath in which embryos are gradually cooled to -30°C and then transferred to two different liquid nitrogen tanks for double banking. Offsite triple banking is available for an additional cost.

Quality control

To assess the viability of the frozen 2-cell stage embryos, one straw from each freezing is thawed and the embryos implanted into the oviducts of the recipient mice. The offspring will be tail clipped to obtain DNA for genotyping, which will confirm the gene of interest has been successfully cryopreserved.