FAQs for Cryopreservation, Cryorecovery, and Breeding Services at JAX
Dolores Garcia Arocena, Ph.D.
At The Jackson Laboratory (JAX), cryopreservation is an integral part of research strategy and the general JAX strategy for managing strains and protecting them. JAX scientists have conducted extensive research in mouse strain cryopreservation techniques to establish highly refined cryoprotectant solutions and optimal protocols for preserving models on diverse genetic backgrounds. These developments are the basis for affordable conservation and colony management practices implemented at JAX, as well as our ability to maintain and distribute more than 11,000 genetically modified mouse lines around the world. Read on to find the answers to our most common questions regarding the cryopreservation, cryorecovery, and breeding services offered at JAX.
FAQs for cryopreservation
Researchers know that maintaining research models as live colonies is financially draining and carries constant potential risks, including disastrous loss due to disease outbreaks, breeding cessation, and facility failures. Cryopreservation techniques are an essential tool for efficient colony management. JAX Cryopreservation Services enable you to not only conserve resources when you are not actively using a mouse model, but also ensure your valuable strains are protected from various disasters, such hurricanes, earthquakes, unforeseen breeding issues or even genetic drift.
Cryopreservation is a very powerful colony management tool that both protects strains from loss and simultaneously ensures that they remain available upon demand (via cryorecovery) for:
- Reinitiation of projects
- Reevaluation of data
- Fulfilling the NIH mandate to share new model organisms with the research community
Can JAX cryopreserve my strains as frozen sperm or frozen embryos?
JAX offers both sperm and embryo cryopreservation services. We can store samples and recover strains for you, as well as support distribution to collaborators of mice or cryopreserved embryos and sperm. We have cryopreserved tens of thousands of strains and recover thousands of strains every year, shipping mice and germplasm globally. If you have an urgent need, JAX has demonstrated the capability to perform large-scale cryopreservation of hundreds of strains at a time for hundreds of different institutions.
Should I try cryopreserving my mice myself or outsource it?
The challenge in cryopreserving sperm is not in the actual cryopreservation. What people find challenging is all that’s required to set up a successful cryopreservation program, whether they are cryopreserving a few strains or hundreds. The cryopreservation of mouse germplasm is straightforward but does require specialized equipment and skills. The proper storage, quality control, and ability to recover sperm successfully requires a much larger investment in equipment and staff. While many organizations have successful cores, the investment in equipment, space, and people to develop the appropriate infrastructure, as well as skills and proficiency, is often prohibitive.
Thus, if considering doing it yourself, it is important to consider not just cryopreservation, but storage and data. If you are interested in doing it yourself, we offer courses as well as a sperm cryopreservation kit. Samples frozen using the kit can be returned to JAX for storage. And if you are interested in outsourcing, always check with any provider, whether a local core or a commercial entity, to make sure they can handle, store, and recover your strains successfully.
Is it best to cryopreserve sperm or embryos?
This is one of the more difficult questions that we are asked. The difficulty lies in your specific circumstances and research. So, we really need to consider two questions.
For strains on pure inbred backgrounds or with few genetic mutations, cryopreserving sperm is the most efficient and least expensive choice. It’s easy to protect your strains from disaster. Our experts perform the service for you, or we can send you a Sperm Cryo Kit.
For strains on unique genetic backgrounds or with multiple mutations or transgenes, embryo cryopreservation may be the best option. Embryo cryopreservation is unmatched in generating experimental mice quickly and has the distinct advantage of recovering the same genetic background and genotypes you need for experiments.
What are the advantages of sperm cryopreservation?
Sperm cryopreservation requires just two males and is quick and inexpensive while achieving reliable recovery of archived sperm. A significant advantage is scalability as millions of sperm are cryopreserved, making it easy to scale the recovery to produce many animals during recovery. You can also recover more mice from sperm than from embryos, so you may be able to produce the amount of mice you need sooner.
How many young males do I need to send for sperm cryopreservation?
We require two males, 8-16 weeks of age.
How do I know that the sperm will be viable in the future?
A best practice is to always make sure that quality control is performed using samples from your strain. There are several options for how to perform quality control. The most basic is to test motility of thawed sperm samples. Knowing that live, motile sperm are present is useful, but motility is not an indicator of fertilizing capacity. A small IVF can be performed to ensure the sperm have retained the ability to fertilize eggs and this approach is the one most often chosen. The sperm can be used to produce live mice and the pups can be genotyped, thus demonstrating that live mice of the appropriate genotype can be produced. This approach is our standard for all publicly available strains in our repository.
How many young males and females do I need to send for embryo cryopreservation of my strain?
Typically 3-4 males, 8-16 weeks of age, and 25-30 oocyte donors to produce 250 cryopreserved embryos. Results will vary significantly among strains and multiple attempts may be required. We have cryopreserved and recovered embryos from thousands of strains and can draw on our experience to help plan projects to ensure success.
How long does it take to cryopreserve sperm and/or embryos?
Projects are typically completed in 12-15 weeks. For sperm, we store 19 straws per strain. Each straw has four aliquots of sperm stored for future use. For embryos, we keep approximately 250 embryos per strain in ten straws for future use.
How should sperm and embryos be stored?
Sperm and embryos need to be stored at very cold temperatures in an environment where thermal stability can be guaranteed. Typically, this requires storage in liquid nitrogen. A best practice is to store samples in at least two tanks that are geographically separated and we store samples in two or three tanks in different locations in order to mitigate risk in case of a tank failure or facility wide issue. JAX also has redundant local and remote alarms systems, redundant power systems, and onsite bulk liquid nitrogen storage to insure that we maintain the integrity of germplasm in our care.
How cost effective is it to freeze down a strain?
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 below, 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. 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).
FAQs for cryorecovery
Can JAX recover my strain from frozen sperm or frozen embryos?
Yes. JAX successfully recovers over 2,500 strains every year for researchers around the world. We can recover your cryopreserved strains for your next project re-initiation, for your collaborators, or cryorecover strains from your collaborators’ cryopreserved stocks.
How long does it take to recover a frozen strain?
Recovery is completed within nine to twelve weeks.
What is most expensive, maintaining a strain I’m not sure if I’ll use it in the next six months, or cryorecover it closer to when I’ll need it?
Cryopreservation costs are typically recovered through cage-cost savings in just months (see the graph above). We have helped hundreds of institutions reduce operating costs by providing an off-site backup of idle research lines. Many investigators choose to spend research funds on data generation, not cage costs.
What is the health status of mice cryorecovered at JAX?
The processes involved in cryorecovering mice using frozen sperm or embryos produce mice that are specified and opportunistic pathogen-free (SOPF), with the highest health status (G200).
For more information regarding cryopreservation and cryorecovery services, visit https://www.jax.org/jax-mice-and-services/cryo-and-strain-donation
FAQs for breeding
Why are my mice not breeding?
Many factors influence mouse breeding success: health status, handling, diet, and the number of hours of light and darkness. It is vital to ensure that they have an adequate dark period for them to mate. Also, keep in mind that many strains are disease models and have limited breeding performance. For more information, check out some tips on general husbandry.
What are the advantages of maintaining mouse colonies at JAX?
- Experience: No one has more mouse colony management experience. Breeding Services staff bring genetics, veterinary pathology, colony, and project management expertise to your project.
- Cost-effective: If you do not have the time, facilities, or other resources to maintain or manage mouse colonies, JAX Services can help. Outsourcing breeding services conserves your limited facility space and technician time, making it a cost-effective option for your in vivo studies. We will maintain, breed, genotype, and ship mice to you as needed, helping you quickly and efficiently achieve your research objectives.
- Maximal health status: You get economical barrier cage housing and receive specific opportunistic and pathogen-free mice without the need for quarantine.
- Fast colony build-up: Jumpstart your timelines by 3-6 months with IVF-assisted rederivation and colony expansion.
- You can access a continuum of integrated services, including blood and tissue collection, genotyping, surgery, histopathology, gene expression, phenotyping, and compound efficacy testing.
What types of breeding projects can JAX do?
JAX can handle any project, large or small. Using JAX® Mice or your mice, we can provide inbred, hybrid, and mutant strain maintenance, chimeric and transgenic founder breeding, multigene crosses, Cre-lox, conditional models, aged mice, special diets, triplet nucleotide repeat models, multiple copy number transgenic models, trisomy models crossed with wild-derived strains, speed congenic line development, and ongoing production of study cohorts for downstream experiments.
Where can I find breeding schemes used to maintain JAX strains?
Find the strain datasheet using the stock # of the strain. Locate the “Technical Support” tab, look for the mating systems we use at JAX, and review the breeding considerations for each strain. Please note that mating schemes are displayed as the female’s genotype(s) x genotype of the male. Keep in mind that the breeding scheme JAX uses may not be the one you need to produce a specific genotype for your studies.
What is the price for breeding mice at JAX?
It depends on which strains you are planning to breed to generate your experimental model, and if they are cryopreserved or live, and if they are good breeders or not. Other variables that factor into the price are the numbers of mice, the genotypes, ages, and sex of the experimental mice needed, as well as how many consecutive shipments you need for your project and the frequency of the shipments.
How long will it take to generate my experimental model developed by breeding two or more strains together?
It depends on which strains you are planning to breed to generate your experimental model, if they are cryopreserved or live, and if they are good breeders or not. Other variables that factor into the timeline are: the numbers of mice; the genotypes, ages, and sex of the experimental mice needed; how many consecutive shipments you need for your project; and the frequency of the shipments need to complete your project.
How do I figure out what breeding schemes to use to generate strains with multiple mutations?
Rather than focusing on the phenotype of the target model you need to make, focus on the genotype of the model you need. This will allow you to map out your breeding better, as well as determine the expected Mendelian ratio of each breeding step, so you can ensure you have enough mice for subsequent rounds of breeding or sufficient mice for your experiments. Specific mating schemes will depend on the genotypes of the experimental mice and controls you need and the viability and fecundity of homozygous mice (watch this JAX On-Demand webinar for more information).
Suppose you don’t have the resources to devote to colony management. In that case, you may leave this up to our experienced team of scientists to figure out the most cost-effective cryopreservation approaches and breeding schemes and monitor your colonies step-by-step until we deliver the experimental mice you need.
Byers SL, Payson SJ, Taft RA, (2006). “Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs). “ Theriogenology. 65(9):1716-26. PMID: 16271754
Kopeika J, Thornhill A, Khalaf Y, (2014). “The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence.” Hum Reprod Update. Dec 17. pii: dmu063. [Epub ahead of print] PMID: 25519143
Ostermeier GC, Wiles MV, Farley JS, Taft RA, (2008). “Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation“. PLoS ONE 3(7): e2792. doi:10.1371/journal.pone.0002792. PMID: 18665210
Wiles MV, Taft RA, (2010). “The sophisticated mouse: protecting a precious reagent.” Methods Mol Biol. 602:23-36. doi: 10.1007/978-1-60761-058-8_2. PMID: 20012390