Cryopreservation of Transgenic Mice: Protect Valuable Mouse Strains and Reduce Costs
Cryopreservation is a powerful tool for colony management, protecting strains from loss while ensuring they remain available upon demand and at the scale needed (see IVF Speed Expansion Services).
Cryopreservation of Transgenic Mice
Cryopreservation is the process of freezing biological material, such as sperm, oocytes, or embryos, at ultra-low temperatures to preserve viability for future use. The Jackson Laboratory pioneered this mouse reproductive technology, enabling the long-term storage and rederivation of live mice from cryopreserved reproductive materials.
We have found that when the frozen material is maintained properly, cryopreserved embryos and sperm remain viable indefinitely. In our more than 30+ years of cryopreserving embryos and 10+ years of cryopreserving sperm, we have not observed a significant decrease in recovery rates with time in storage.
Embryo and Sperm Cryopreservation of Mouse Models
Both embryo and sperm cryopreservation are well-established, validated methods for preserving and rederiving live mouse lines. Neither method is inherently superior; the best choice depends on several factors:
Strain Characteristics: Sperm cryopreservation involves freezing only the male gametes. To recover the line, the frozen sperm is used to fertilize oocytes from wild-type females. This method is best suited for strains carrying a single-gene mutation and maintained on a common inbred background where genetically matched oocyte donors are readily available. Maintaining the genetic background is important, as it can influence phenotypic expression. In contrast, for strains on a hybrid or unique genetic background, or those carrying multiple unlinked mutations, embryo cryopreservation is best. Embryo cryopreservation preserves the full genetic background and genotype, avoiding any potential recombination or genetic changes that can occur during recovery breeding and impact phenotype.
Experimental Objectives and Timeline: Because sperm cryopreservation only preserves the paternal genome, recovered offspring are generally heterozygous for the mutation. Additional breeding is often needed after cryorecovery to generate homozygous or compound genotypes (e.g., double/triple knockouts), which can extend timelines. Embryo cryopreservation, however, preserves the full genotype, both maternal and paternal contributions, at the time of freezing. This allows for direct recovery of the exact original genotype, including homozygotes and complex or multiple unlinked mutations. However, this may require more upfront breeding to generate the desired genotype you want to freeze.
Cost and Resource Availability: Sperm cryopreservation is generally more cost-effective upfront and typically requires only two males. Embryo cryopreservation, however, requires both males and females. If the females respond poorly to superovulation, a higher number may be needed to produce sufficient embryos for freezing, which can increase resource demands.
Advantages of Mouse Model Cryopreservation
Animal researchers know that maintaining research models as live colonies is financially draining and carries constant potential risks, including disastrous loss of entire strains or significant delays in research due to disease outbreaks, breeding cessation, and facility failures. For example,
In 2012, Hurricane Sandy flooded NYU Langone Medical Center, wiping out over 3,000 mouse lines and halting the work of 50 researchers. The financial impact was in the tens of millions. More devastating, however, was the loss of valuable lines – some researchers had no cryopreserved backups, meaning decades of work were wiped out in a single night, with no possibility of recovery.
In the 1970s, a researcher was relocating 15,000 mice across states to his new laboratory. Despite the move being successful in transport, breeding significantly slowed. Eventually, the researcher lost 25 unique and irreplaceable strains due to breeding cessation in his new lab following the move.
The lesson: Cryopreserve any mouse strains that are critical to your research program, especially those you created yourself or that would be difficult to reacquire. If your colony is affected by breeding errors, genetic drift, phenotype loss, or a pathogen outbreak, AND you have the strain cryopreserved, you can recover the original strain and resume your work with minimal delay.
Cryopreservation also enables long-term storage of valuable or currently unused genetic lines without the cost and effort of continuous breeding. It also allows for rapid reestablishment of colonies at scale when needed, ultimately reducing the number of animals used and lowering overall costs.
Figure 1. Cost-benefit of cryopreserving versus maintaining live colonies of low use research strains.
Cryopreservation costs are typically recovered through cage-cost savings in just months. Colony costs depend on the number of boxes maintained and the cage cost/day. The cost to maintain a heterozygous colony that requires frequent genotyping ranges from a few thousand dollars to over $10,000 per year.
As shown in the graph above, even in a scenario with low per diem rates, the cumulative cost of maintaining a colony increases significantly faster than the cumulative cost of cryopreserving a strain. Note that this strategy is cost-effective even if periodic recoveries and subsequent short-term maintenance of a live colony are needed (e.g., between years 2-3 and 6-7, as illustrated).
In recent years, and more acutely in recent months, significant federal funding cuts have increased pressure to reduce operating expenses. As a result, many institutions are turning to outsourced cryopreservation and recovery services. This shift eases the burden on investigators and core facilities, particularly when working with genetic backgrounds known to respond less reliably to the cryostorage and reanimation process (Wiles et al. 2010).
Outsource cryopreservation services?
You may ask yourself: Should I try cryopreserving my mice myself, or outsource it? Whether using a CORE facility or a trusted vendor like JAX, we highly recommend outsourcing. If biopreservation is not done correctly, a strain may be lost. Cryopreservation using suboptimal conditions can result in:
Extensive damage to cell membranes
Alteration of the functional and metabolic status of the cells and mitochondria
Increase in DNA single-strand breaks
Keep in mind that the extent of this damage can vary by the methods employed (Kopeika et al. 2014) and may be genetic background-sensitive. Generally speaking, the expense of training, operating, and maintaining in-house cryopreservation facilities is not cost-effective for most small or medium-sized academic or biotech organizations.
Research-based insights into affordable management of mouse models
Over the past decade, scientists at JAX have conducted extensive research in mouse strain cryopreservation techniques to establish optimal cryoprotectant solutions and optimal cooling and warming protocols for preserving research mice on diverse genetic backgrounds (Byers et al. 2006, Ostermeier et al. 2008, Wiles et al. 2010).
These developments are the bases for affordable conservation and colony management practices implemented at JAX, as well as our ability to reliably distribute more than 10,000 genetically modified mouse lines around the world. Using our pioneering cryopreservation techniques, JAX has cryopreserved more than 4 million embryos from 20,000 strains, and we routinely recover successfully more than 2,000 strains a year. Moreover, JAX has supported hundreds of institutions in the large-scale cryopreservation of over 100 of their idle research strains.
JAX® has a range of Cryopreservation and Recovery Services to meet different research needs. Contact us at micetech@jax.org and our team can assist you in identifying the best solution for your projects.