Transgenic Resources Program

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EMBRYO CRYOPRESERVATION

Embryos intended for freezing are collected from time-mated females. The hormone injections and matings take place in your colony by your lab to prevent transmission of disease into the K-wing transgenic colony. We collect the embryos, freeze (including a test straw) and store them in 2 separate LN2 tanks for added security. The upper limit handled on a single day is the number of embryos generated by 15 donors (up to 2 separate lines). We provide the hormones for superovulation, and you provide all of the mice.

The first step in the process is to collect an inventory of your mice to determine the best way to go about generating embryos. We are willing to provide advice. In general, male mice breed well until 8 to 12 months of age (with rare exceptions) and females breed well from 7 weeks of age with slow loss of fertility after 12 weeks. Because females are not optimally fertile for very long, the procedure runs more smoothly and efficiently if immature (3 week old for most strains) females are purchased to be superovulated and mated to transgenic males. Adult females can also be superovulated, but less reliably. Matings are conducted in the ratio of one female to one male. Males are housed individually and the female is introduced into the male's cage immediately after the hCG hormone injection. All of the females should be checked for plugs the following morning and removed from the male's cage. It helpful to keep a record of each male's plugging history.

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SUPEROVULATION

E0.5 = the morning after hCG (day of copulation plug ),
E2.5 = the morning of 8-cell embryo collection.

For example, if embryos are to be collected on Thursday :
PMSG is given Saturday
hCG is given Monday
E0.5 is Tuesday
E2.5 is Thursday

The E2.5 pregnant females should be delivered by 9:00 AM.

Alternatively, adult females can be used that are mated without superovulation and the embryos collected on E2.5 or E3.5. Exogenous hormones tend to cause the release of oocytes that have an immature zona, making them less able to transition from the oviduct into the uterus intact. Zona-free embryos cannot be frozen. This problem does not occur with naturally mated females, or E2.5 embryos, which are still in the oviduct.  Significantly fewer embryos are collected from naturally mated adult females compared with superovulation of immature or mature females. However natural mating may be preferable if hormone administration is difficult to schedule, maintaining a homozygous line is important, or preserving embryo quality becomes an issue.

We collect the E2.5 embryos from the oviduct side of the utero-tubal junction. Embryos will be selected for cryopreservation by assessment of normal developmental progression and an intact zona pellucida. They will be washed through a trypsin-EDTA solution to remove any contaminating pathogen sensitive to trypsin that may have adhered to the zona. We will load up to 30 embryos per straw (enough for 2-3 transfers upon thaw). Once the embryos are placed in the cryoprotectant, there is a half-hour before they are placed at -7°C and the freezing process begun. We freeze at -0.3°C/min. down to -33°C, then -0.1°C/min. down to -35°C. There are several protocols for freezing rate and cryoprotectant. For instance, The Jackson Lab freezes down more quickly. A slower freeze will not harm the embryos, and may actually be beneficial. The cryoprotectant is 1.4 M glycerol. The straws are loaded with an excess of 1M sucrose separated from the embryos by an air bubble. This is useful on thaw, as straws need only be shaken to drop the embryos into 1M sucrose for removal of excess cryoprotectant. This method was developed so that no special media need be made upon thaw. There is no impact on survival due to method of thaw, whether by step-wise cryoprotectant removal or the 1-step method described above.

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SUMMARY OF IMPORTANT DETAILS

1. Take an inventory of the mice scheduled for cryopreservation (you'll need up to 15 transgenic males).
2. Order embryo donors 2 weeks prior to the cryopreservation day.
3. PMSG and hCG are available from Shoshana Maslan in the K-Wing vivarium.
4. Deliver the mice by 9:00 AM on day E2.5

It is important to be aware that many factors can influence the number and viability of embryos collected: weight, light cycle, hormone dosage, transgenic alterations, and genetic background strain. Ideally, female donor mice should be hormoned at 4 weeks of age and weigh about 14-16 grams. In addition, it is vital to use experienced, proven male breeders.

GUARANTEE AND CHARGES

Cryopreservations are charged on a per session basis, with a maximum of 15 donor females per session. A test straw will be thawed immediately after freezing in order to provide information on the survival and developmental potential of your stored embryos. An 80% survival rate is expected upon thaw. If we experience a failure as determined by < 50% survival the session will be repeated at no charge. The cryopreservation fee covers storage in LN2 for 5 years. At the end of this period, you may choose to purchase another 5 years of storage, move your embryos to your lab, or request in writing to dispose of the cryopreserved embryos.

EMBRYO REDERIVATION

As a completely separate service, we can thaw and transfer embyros to pseudopregant recipients. The cost of embryo rederivation includes thaw and transfer of one straw of embryos to time-mated pseudopregnant females. You will also be responsible for per-diem cage costs. If the recipients do not become pregnant for any reason, we will repeat the procedure at no additional cost. The recipients will be housed in the K-wing quarantine room with routine monitoring until they give birth. After the pups are weaned, the recipients will be tested for pathogens before the mice are transferred to your colony.

Click here for rates.