RRC-TPS: Cryopreservation

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INTRODUCTION Cryogenic freezing of mouse embryos and sperm provides security against loss of valuable mouse lines due to disease, environmental disaster, and human error; it provides a cost-effective method to store lines not currently in use; and it provides a convenient way to ship important mouse strains to other investigators world-wide.

Two-cell embryos are cryopreserved using a vitrification method, performed by briefly exposing the embryos to a cryopreservative containing DMSO and sucrose before plunging them into liquid nitrogen. Upon thawing and transfer to surrogate mothers, an average of 30% of cryopreserved embryos on a C57BL6 background develop to pups. Variability in the success rate with other strains may be related to genetic background. The embryo vitification service includes a test-thaw of one vial of frozen embryos for each line frozen. Embryos will be thawed 1-2 weeks after freezing and transferred to surrogate mothers for development to term to verify recoverability. At this point the investigator will be notified as to the success of the procedure and the decision to sacrifice the line may be made. At the investigator's discretion the test-thaw will not be performed, with a reduction in fees.

Sperm are cryopreserved by freezing in a solution of raffinose and skim milk. It is a simple procedure requiring only 1 male for sperm collection. Recovering the line from frozen sperm is performed by in vitro fertilization (IVF). The success rate of IVF is variable between strains: Outbred and F1-hybrid strains typically work very well, but inbred strains and strains of mixed genetic background can be problematic. Even though recent modifications to the sperm cryopreservation procedure have improved the viability of thawed sperm, particularly for the popular, but historically difficult C57BL/6 strain, the fertilization potential of thawed sperm cannot be guaranteed.

Fees for storage of cryopreserved embryos and sperm in LN2 are charged at the rate of $20.00/strain/quarter. The quarterly fee for each strain will appear on RRC billing statements in January (for the months of Oct-Dec), April (for the months of Jan-March), July (for the months of April-June), and October (for the months of July-Sept).

In addition to cryogenic freezing, we also offer services to recover imported cryopreserved lines.

INSTRUCTIONS

  1. The investigator must open both an RRC account and a BRL account as needed. The BRL account will be used to purchase and house any mice required to complete your project. The investigator must also review our User Policies.
  2. If live mice will be generated, for example as a result of projects to recover cryopreserved lines, the investigator must submit a UIC Animal Use Protocol for approval by the Animal Care Committee (ACC) before initiation of this project.
  3. The investigator must complete the an Application (contact TPS Director) for cryogenic services.

Mating schemes to generate 2-cell embryos for cryopreservation. The approximate goal for each line is to freeze at least 200 embryos @20 embryos/vial. To obtain this many embryos in a single day females must be induced to superovulate prior to mating. Due to variability in both response to the superovulation regimen and breeding performance it may take more than one attempt to collect enough embryos. If additional attempts to obtain sufficient embryos are requested, all animal fees and 50% of the Cryopreservation Production Fee will be charged per attempt. Depending on the mating pairs chosen, not all the embryos will necessarily carry the gene (transgene or knockout allele) of interest. For example, in the case of heterozygous mutant males mated with wildtype females, only 50% of the resulting pups will carry the gene of interest; this means that after freezing and thawing, the pups derived from frozen embryos will have to be genotyped and additional breeding performed to reconstitute the line.

The following is a guideline only; it is important to discuss specific details for your particular line with the TPS Director well in advance. Depending on where your mice are housed, special arrangements may have to be made.

  • You must provide us with 10-12 heterozygous or homozygous transgenic (Tg) or knockout (KO) males at 8 weeks to 8 months of age. When selecting males, make sure they have not been used for breeding for at least 2 weeks and have been housed individually (1/cage) for about two weeks. We will also need 10-12 embryo-donor females of superovulatory age (varies with strain). Wildtype females are typically purchased for this purpose and will be charged to your BRL account. Females from your transgenic breeding colony may in some cases be used, but keep in mind that they may not respond ideally (if at all) to superovulation.
  • Superovulation will be induced by injecting the gonadotropins, PMSG (pregnant mare's serum gonadotropin), followed by HCG (human chorionic gonadotropin) 48 hrs later. Superovulated mice will be mated with the males immediately after the second injection. Two days later, two-cell embryos will be collected from plugged females and frozen.

Sperm cryopreservation. If you choose to freeze sperm, you must provide us with 1 or 2 males of the desired genotype. To recover pups at a later date IVF will have to be performed. As discussed above, the success rate of IVF with frozen sperm is strain-dependent.

Recovering mice from frozen embryos. Following thawing, embryos will be transferred to pseudopregnant foster mothers for development to term. Pups will be turned over to the investigator at weaning. Depending on the genotype of the of the parents of frozen embryos, pups may have to be genotyped and additional breeding performed to reconstitute the line. All animal purchases/per diem will be charged to your BRL account. We cannot assure success with embryos cryopreserved outside our facility.

Recovering mice from frozen sperm (IVF). For lines preserved as frozen sperm IVF is used for recovery. IVF will be performed using oocytes collected from wildtype females. The day following IVF, two-cell embryos will be transferred to pseudopregnant foster mothers. The resulting pups will be turned over to the investigator at weaning. Depending on the genotype of the sperm-donor, pups may have to be genotyped and additional breeding performed to reconstitute the line. All animal purchases/per diem will be charged to your BRL account. These include mice used for oocyte-donors, as well as females used to establish pseudopregnancy in surrogate mothers. As discussed above, the success rate of IVF with frozen sperm is strain-dependent; as such the fertilization potential of thawed sperm cannot be guaranteed.

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