Embryo Cryopreservation

Due to the inherent value of genetically altered mouse strains, we offer cryopreservation of individual mouse strains housed at UNMC. Strains will be cryopreserved in the order in which requests are submitted. Please read the following information carefully to understand the benefits and limitations of cryopreservation.

Provision of animals

Investigators should contact the cryopreservation service once they have ascertained that they have sufficient numbers (see below) of fertile males and, if needed, female mice. The investigator should provide a minimum of 10 males aged 2 to 6 months. Depending on strain background requirements, the investigator may supply females aged 3-4 weeks or MGECF will purchase the correct background strain from an approved SPF vendor. Mice will be transferred to MGECF and per diem will be covered by the core facility. Females will be superovulated, mated with males and embryos harvested and cryopreserved. This procedure will be repeated until 300-500 embryos are cryopreserved (see below).

Cryopreservation

Gestational day 2.5 embryos are cryopreserved in straws and stored in liquid N2. A set of control embryos will be harvested and cryopreserved on the same days as the mutant lines. The first and last straw will be subsequently thawed and allowed to mature to the blastocyst stage to demonstrate viability of the line with the assumption that all embryos frozen between the first and the last straw will behave similarly.  For an additional charge, cryopreserved embryos can be thawed and transferred to foster females to demonstrate viability of the line. The thawed embryos are transferred into pseudopregnant females. Females are dissected at late gestation and can be used to verify the genotype if desired. Upon completion of cryopreservation, we will notify you.
If homozygous embryos are preserved, we recommend the storage of at least 300 embryos, in addition to those frozen and thawed to ascertain viability. If heterozygous embryos are preserved, we recommend the storage of approximately 500 embryos.

Other Considerations

The success of cryopreservation is dependent upon the fertility of the animals used and the response of the females to superovulatory hormones. The most efficient strategy is to use 3-4-week old females of the appropriate strain supplied by a commercial vendor. However, it is appreciated that in many instances investigators must work within strain background constraints that preclude this option. In those instances, we will cryopreserve embryos on the appropriate genetic background, if it is possible to do so; realizing that more attempts and more mice (and hence higher costs) will likely be required.

There will be a yearly liquid N2 fee to maintain the embryos in liquid N2 tanks in our facility. You will receive a statement in December of every year, detailing the charges for your cryopreserved lines.

  1. Submit the information about the mouse line to be cryopreserved using the Cryopreservation Form.
  2. Fill out FORM. Be sure to include a cost/center number for billing, and your animal protocol number. Submit the form and the Core lab will receive the electronic forms, as well as you will receive an electronic copy.
  3. Provide the facility with proven breeder males of confirmed genotype (10 males aged 8 weeks to 6 months). If it is absolutely critical to freeze homozygous embryos, you must at that time also supply enough females aged 4 to 6 weeks for at least 3-4 rounds of superovulation.  Each round requires 20 females to be bred to 10 males or 40 females for 2 rounds.
  4. You will be notified upon the successful completion of our cryopreservation procedure.

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