Embryo cryopreservation is an efficient, reliable, and cost-effective means of safeguarding and maintaining stocks of critically GEM resources. The core will coordinate with PIs by the mouse age to schedule the cryopreservation date. The core recommends freezing 150 to 500 embryos for each line of mice. This usually requires freezing embryos more than one time.
What Will Happen
- Investigator will submit a request form through iLabs, filling out detail information on male and female mouse numbers and ages, and any known reproductive defects. An account and IACUC protocol number must be provided at this time. The Investigator IACUC protocol must be approved for all procedures performed in the user laboratory.
- The core will review investigator provided information and approve the request. The core will schedule the cryopreservation according to the female mouse age and coordinate with the investigator.
- Investigators will complete steps 3-10. Investigator prepares 8-10 adult males and 8-10 young female mice at age of 3-5 weeks with appropriate genotypes.
- One week before the scheduled cryopreservation date, pick up 1 tube of M2 media, 1 tube of PMSG, and 1 tube of HCG from the GERM Core. Note: Bring an ice bucket to put the hormones in and put them in a -20C freezer when you get back to your lab. Dilute the hormones right before you go to give the injections.
- PMSG is given at 10 a.m. three days before the scheduled rederivation date (e.g. Tuesday for cryopreservation on Friday). Dilute the PMSG with 950 ul of 0.9% saline and administer 0.1cc, i.p. per mouse.
- HCG is given 47 hours after PMS at 9 a.m., one day before the scheduled rederivation date (e.g. Thursday for cryopreservation on Friday). Dilute the HCG with 950 ul of 0.9% saline and administer 0.1cc, IP per mouse.
- Place the females with the males immediately after administering HCG.
- The morning following the HCG injections, the females are removed from the male cages.
- The morning following the HCG injections, the females are removed from the male cages and oviducts are collected into M2 media provided by the GERM Core (a protocol will be provided).
- Label the tubes with the request number generated by iLab. Deliver the oviducts to R119A in the ABBR and leave in the biosafety hood no later than 9 a.m.
- The core will dissect, digest and collect all oocytes from the oviducts and cryopreserve all fertilized 1-cell embryos.
- The core will store the cryopreserved embryos in our liquid nitrogen tank up to one year and the PIs need to send the cryo stock of their mouse lines to a commercial company for long term storage.
What is Expected
- The number of embryos that could be cryopreserved depends on the number of embryos we could collect from investigator-delivered oviducts. We freeze all embryos at 30-40 embryos per straw.
- Quality control is completed by thawing about 10 embryos from the batch of cryopreserved embryos processed on the same day, not necessarily each request, and check under microscope to assess viability rate. A rate of over 60% viability passes our QC.
- If there are no fertilized embryos collected, the core will charge 40% of cost. Investigators can repeat the cryopreservation at 100% cost.
- If a reproductive defect is known for the specific GEM model, the total number of embryos collected could be low. In such an instance, additional cryopreservation attempts will be done at 100% cost.