Embryo cryopreservation is an efficient, reliable, and cost-effective means of safeguarding and maintaining stocks of critically GEM resources. The core will coordinate with Investigator by the mouse age to schedule the cryopreservation date. The core recommends freezing 150 to 500 embryos for each line of mice. This number usually requires multiple rounds of producing embryos for freezing.
What Will Happen
- Investigator will submit a request form through iLab, 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 Investigator laboratory.
- The core will review investigator provided information and approve the request. The core will schedule the cryopreservation according to the appropriate mouse age and coordinate with the investigator.
- Investigators will complete the following steps to prepare embryos:
- a. Investigator will prepare 8-10 adult males and 8-10 young female mice at age of 3-5 weeks with appropriate genotypes.
- b. 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.
- c. PMSG is administered via intraperitoneal injection (IP), at 10 a.m. three days before the scheduled rederivation date (e.g. Tuesday for rederivation on Friday). Dilute the PMSG with 950 ul of 0.9% saline and administer 0.1cc IP per mouse.
- d. HCG is administered via IP injection 47 hours after PMSG, at 9 a.m. one day before the scheduled rederivation date (e.g. Thursday for procedure on Friday). Dilute the HCG with 950 ul of 0.9% saline and administer 0.1cc IP per mouse.
- e. Place the females with the males immediately after administering HCG.
- f. 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.
- g. Label the tubes with the iLab request number. Deliver the oviducts to ABBR R119A and leave in the biosafety hood at room temperature 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. The Investigator will need to send the cryo stock of their mouse line(s) to a commercial company for long-term storage.
What is Expected
- The number of embryos for cryopreservation depends on the number of embryos collected from investigator-delivered oviducts. We freeze all embryos at 30-40 embryos per straw.
- Quality control for the freezing process is completed by thawing a batch of wild-type cryopreserved embryos processed alongside the investigator request on the same day, and visually checked 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.