Archiving best practice

Archiving a strain through cryopreservation can be used to provide a back-up to production colonies, minimise genetic drift over long periods of time, reduce the need for tick-over/maintenance colonies and preserve valuable models, especially those for which there is no present need. 

This best practice guidance covers:

Choosing an archiving method

When choosing an archiving approach, it is important to consider technical requirements, financial costs and the number of animals required for each method (see Table 1), as well as the time it takes to recover a cryopreserved strain (see Table 2). All archiving methods have well established protocols (see Cryopreservation protocols on the Infrafrontier website) and both embryos and sperm can be easily transported in a liquid nitrogen dry shipper (although frozen sperm can also be sent on dry ice).

 

Table 1. Considerations for selecting an archiving method

 

Embryos

Sperm

  Superovulation with natural mating Superovulation with IVF*  

Number of animals required

10 – 20 females plus 3 – 5 stud males. 5 – 10 females plus 1 – 2 males for sperm.

2 – 4 males.

Cost and equipment or techniques required

Freeze

High cost – requires a controlled-rate freezer.

Lower cost alternative – embryo vitrification.

High cost – requires specialised incubator and controlled-rate freezer.

Lower cost alternative – embryo vitrification.

Low cost – simple and cheap freezing method with no special equipment required and minimal training.

Recovery

Low cost – embryo transfer of thawed embryos.

Low cost – embryo transfer of thawed embryos.

High cost – IVF requires specialised incubator followed by embryo transfer.

Efficiency

Low efficiency – use of large number of animals, some strains do not superovulate well.

Medium efficiency – 6 to 12 animals are needed on average to preserve a strain.

High efficiency – fast and generates large volumes of embryos on recovery.

Genotype recovered

Any genotype.

Heterozygous or wild-type only.

Recovery method

Embryo transfer (surgical or non-surgical).

IVF followed by embryo transfer (or on occasion artificial insemination).

Special requirements

Breeding to generate stock for cryopreservation.

Oocyte donor.

Quality control

Before freeze

  • Confirmation of genotypes of stock animals before cryopreservation.
  • Confirmation of genotypes of stock animals before cryopreservation.
  • Check for sperm motility and volume at point of cryopreservation.

 

After freeze

  • Test thaw one straw and culture to blastocyst stage (for strains known to have issues recovering animals post cryopreservation, an embryo transfer and live pups can be obtained to confirm viability, but this is not recommended as routine practice).
  • Genotype embryos after thawing.
  • Test thawed sperm in IVF with 1 female.
  • Ideally genotype resulting embryos.
  • Freeze remaining embryos if possible.

* New technologies/reagents are becoming increasingly available to increase the efficiency of superovulation, for example the use of inhibin antisera to increase the number of eggs (hyperovulation). These technologies could reduce the number of mice needed to produce enough embryos for IVF/freezing, however there can be associated welfare concerns. 

Laboratories that are unable to set up their own in-house cryopreservation facilities might like to take advantage of the free-of-charge (shipping costs only) archiving services offered by members of the European Mouse Mutant Archive (see the European Mutant Mouse Archive website). The European Mutant Mouse Archive supports the mouse research community across Europe and beyond.

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Key considerations when cryopreserving a strain

Age of animals

  • Males should be 10 – 24 weeks of age ideally. Males can be older but there is a decreased chance of obtaining good quality sperm. Sperm can be frozen from eight weeks of age, but sperm volume is often lower, especially in more complex crosses and some backgrounds.
     
  • Females should be three to six weeks of age ideally. Females can be used up to ~24 weeks, but fertilisation/recovery of embryos will decrease significantly with age, requiring the use of more animals.
     

Record keeping

  • It is important to keep accurate and comprehensive strain notes on allele, background and phenotype (see mouse passport guidelines for recommendations).
     
  • Keep a record of the freeze/thaw method used, as this may change over time.
     
  • Consider freezing some tissue in case you want to genotype in future.
     

When to freeze

  • On arrival/creation of a strain, freeze sperm from one to two males.
     
  • When backcrossing a strain, freeze sperm from one to two males, every three to five generations.
     
  • For complex breeding consider the value of freezing intermediate breeding steps.
     
  • Once the final product is ready (e.g. multi-allelic, backcrossed), freeze embryos and sperm as per Table 2.
     

Storage

  • Ensure your samples are split across two banks, and preferably two sites.
     
  • Liquid phase is recommended for storage given the small volume in straws.
     

Quality control measures

  • It is important to always perform quality control checks before and after freezing, following the recommendations in Table 1.

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Timelines for archiving methods

Table 2 has examples of the number of males and embryos generally recommended to safely archive a strain without keeping any animals in a live colony. It should be noted that freezing sperm will allow the recovery of most strains and alleles, but that this approach requires longer to recover the strain in its experimental state for multi-allelic strains.

Table 2. Animal numbers and expected timelines for archiving different types of GA strains**

Example of strain required

Number of embryos

Number of males

Estimated time for cryopreservation

Estimated time for recovery of adults from sperm

Single allele GA strain, heterozygous.

0

2

If a supply of wild type females is available, then time to freezing is only dependent on the age of the males.

Freezing is possible when males reach 10+ weeks of age.

Adults available in 10 weeks.

Single allele GA strain, homozygous.

50 – 100

2

6 – 10 weeks to breed sufficient females for IVF/natural mating, or longer if a male needs to age for IVF or repeat rounds of breeding are required.

20 weeks minimum – very dependent on the genotyping protocol, likely closer to 30 weeks.

Multiple allelic strain, heterozygous.

50 – 250

2 – 4

If a supply of wild type females is available, then time to freezing is only dependent on the age of the males.

Adults available in 10 weeks.

 

Multiple allelic strain, homozygous.

100 – 250

2 – 4

Potentially up to 20 weeks to breed sufficient females for multiple rounds of IVF. (More complex strains and those on other backgrounds often respond poorly to superovulation).

Dependent on strain complexity, this could take up to a year.

** Examples are based on C57BL/6J substrains, requirements and timelines may vary for other backgrounds.

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