Cryopreservation

Initiating the cryopreservation project

1. Complete a cryopreservation service request form. The form represents a service agreement, outlining the terms and conditions of the service and defines the details of project cost. This needs to be signed by the principal investigator prior to commencement of work.
2. Complete the technical information sheet at the end of the Cryopreservation service request form which provides the details of the strain and its location within the University.
3. Raise a Puchase Order for the project service fee (see below) and include this in the technical information sheet (For WTCHG-administred grants, only the grant code is necessary)
4. Send the completed form to the following email address: transgenics@well.ox.ac.uk

Breeding strategy for the collection of embryos

• We strongly recommend that embryos should be generated for cryopreservation by mating genetically modified studs (heterozygous or homozygous) with wild-type females mice. Embryo donors are typically superovulated as part of the embryo harvest procedure and the efficiency of this superovulation process depends critically upon the strain and the age of the females. The use of wild-type females bought at an appropriate age maximizes the efficiency and speed of the cryopreservation project by maximizing the embryos yield.
• If heterozygous males are used as studs with wild-type females, a larger number of embryos will be cryopreserved to compensate for the mix of genotypes.

• There are some cases where it would be advantageous to cryopreserve homozygous embryos or embryos which harbor multiple transgenes or modified loci. This would necessitate the use of appropriately aged genetically modified females which will be mated with genetically modified studs. The superovulation process works efficiently with immature mice and subsequently, breeding boxes to generate the required females will be needed. If the efficiency of embryo production is low (which is frequently the case with genetically modified in-bred females), the cryopreservation process may require multiple sessions which thus require several experimental groups of appropriately aged females.

  For single locus homozygous strains, it is recommended to use these studs with wild-type females to obtain heterozygous embryos and to breed back to homozygosity once the strain is revitalized.

  Where genetically modified females are to be used, it is essential to discuss this matter with the Wellcome Trust Centre for Human Genetics Transgenic Core prior to project commencement.

Minimum numbers of animals required to commence a cryopreservation experiment

Where wild-type females are used as embryo donors:

• 5 stud males of appropriate genotype and of breeding age (preferably of proven fertility, i.e. ex-breeders)
• Wild-type females required for the project will be purchased directly by the transgenic core

Where genetically modified females are used as embryo donors (e.g. homoyzgous or lines harbouring mutliple transgenes)

• 5 stud males of appropriate genotype and of breeding age
• At least 4 groups of at least 5 immature (3-4) week old females which will frequently need to be bred in the quarantine facility. The costs involved in breeding these mice are not included in the project cost and will be recharged to the group in the monthly cage cost bill

The cryopreservation can commence

1. Embryos will be generated from the requested strain by mating male studs with superovulated females. The embryos will be cultured to the two cell stage, followed by cryopreservation and storage in two independent locations.
2. The breeding boxes for embryo production will be housed in your space allocation and the cage charges incurred will be recharged to your group via the usual BMS recharge system.
3. Depending on the mix of genotypes present in the resulting embryos, between 100 and 200 embryos will be cryopreserved per project.
4. For viability testing of the frozen embryo stocks, a choice of service is provided: an in vitro test, where a sample of frozen embryos is thawed and viability assessed by monitoring cleavage events in culture, and an in vivo test, where a sample of frozen embryos are thawed and then transferred into a pseudopregnant female to assess the ability of the recovered embryos to develop to term. The more intensive in vivo test service is recommended for critical strains which cannot be re-sourced elsewhere.
5. Once the thawing tests have been performed, we will feed-back on the viability and advise whether further embryos should be generated. Please wait for our viability report before culling you active colony.
6. Currently, there is no fee for storage. If policy concerning sample storage changes, additional fees will apply

Project charges

Costs are calculated based on a service fee plus the real cost (including delivery) of wild-type mice (e.g. embryo donors) which have to be purchased for the project. The number of sessions (i.e. rounds of superovulation and embryo harvest) required to secure the cryopreservation of a line is dependent upon the type of strain, making it difficult to specify a fee in advance. The cost of wild-type mice per cryopreserved strain is likely to be in the region of £150-250.

On completion of a project, groups will be asked to raise an additional Purchase Order to meet the real cost of mouse purchases for this project.

In addition, please consider that during the cryopreservation project, the housing of the genetically modified studs (and potentially female mice) used for embryo production will be charged back to your group as part of the normal BMS recharges.

Service fee (in vitro thaw): £420(WTCHG held grants) ; £560 (non-WTCHG held grants)

Service fee (in vivo thaw): £950(WTCHG held grants) ; £1280(non-WTCHG held grants)