The cryopreservation of embryos is becoming increasingly more popular due to a number of advantages it has over maintaining a colony. Some of its advantages are, large financial savings on cage costs, prevention of genetic drift, less time spent on colony maintenance, and a pathogen free backup in the event of contamination.
To begin a cryogenic project at the facility, investigators are requested to provide us 10 male mice of the line to be frozen and between 30-50 females of that line, or other strain desired. The number of females will be determined on an individual basis, dependant on the lines breeding record. The Transgenic core will do breeding to obtain the necessary animals, if mice are not available. This breeding will be an additional charge to the investigator. Following is the timeline for a cryopreservation project.
Embryo Freezing Timeline
|Time depends on colony size||Breeding of mice to obtain 5-10 male and approximately 20-30 females preferably at 5-6 weeks of age.|
|3 days:||Hormone priming of donor females|
|1 day:||Check mating plugs of donor females|
|1 day:||Embryo development to 2 cell stage|
|1 day:||Cryopreserve embryos|
* Each round of cryopreservation takes 1 week. The number of rounds needed for a project will vary depending on breeding performance of the background strain. Feel free to contact us in regards to the animals needed for your specific project.
Responsibilities of the Investigator:
– Provide the Facility with 10 males (preferably homozygote mice).
– If these mice are not provided and the Facility must breed, the investigator is responsible for related charges.
– Animals will be transferred to Transgenic Facility protocol, but will remain under P.I account for cage charges by LASC.
-Investigators are asked to create a back-up of their line, (ex. send animals to collaborator or keep one breeding cage) as there is never 100% guarantee in the cryopreservation/storage process.
Responsibilities of the Facility:
– Material and labor needed for the cryopreservation of 250 or 500 embryos. (to be specified by the investigator).
– When provided with homozygous males, the Facility will order the females (10 per day) and will pay the cost for housing.
– When it is necessary to breed for freezing, the Facility is available do so at charge to the investigator.
In Vitro Fertilization (IVF)
Goals: The 2 primary goals of IVF are to rescue lines, which for one reason or another stop breeding, or to re-derive a line, which was frozen down using sperm.
Day 1: PMS injection
Day 3: HCG injection
Day 4: Isolate sperm (or thaw) and embryos. Perform IVF
Day 5: Transfer all fertilized (2 cell) embryos into a Psuedo-pregnant female.
Day 26: Litters are born
Day 47: Litters are weaned
Day 61: Animals are tested for pathogens and transferred to investigator.
Animals: Investigators will need to supply the Transgenic Facility 1-2 males, from which fresh sperm will be isolated and used for IVF, or with 1-2 straws of frozen sperm. Fresh sperm works better and ideally males should be 2-5 months old and proven good breeders. The transgenic core will provide the embryos, which will be obtained from WT females.
Success: IVF is a very tricky procedure, and its success depends on many factors. The largest determinant is strain or sub-strain used. There is great variation of success based on the strain of the animals used as well as the age and breeding capabilities. This variation ranges between 90% efficiency in some strains to <5% in others. The Transgenic Core therefore cannot guarantee the successful re-derivation or rescue of your line. The transgenic core is well experienced, and will do its best, including a second attempt, if the first round of IVF fails. The charges collected cover the IVF, the animals and per diem cage costs, as well as all supplies and labor. If the In Vitro Fertilization is not successful in your particular strain, there are still other options that the core can direct you to in order save your line.
Genotyping: Since the sperm will fertilize a wild type egg, your homozygote transgenic line or knockout line will become hetrozygote after IVF, needing cross breeding to re-achieve homozygocity.