We recommend freezing 150 to 500 embryos for each line of mice. This usually requires freezing embryos more than one time. (Note: Each time you freeze a line, a MEMS request form must be filled out. Each request should have one line associated with it, no multiple lines on the same form). Cryopreservation is accomplished by the following:
1. Superovulating 3-5 week old females with hormones (PMS and hCG).
2. Placing the females with males.
3. On the scheduled day, euthanize the female mice by cervical dislocation under anesthetic. Collect the reproductive tracts.
4. Euthanizing the female mice.
5. Isolating the reproductive tracts.
6. Collecting the fertilized one cell embryos.
7. Freezing the 1-cell embryos.
The best response to superovulation is achieved using 3 week old females. Wild type females can be ordered from a vendor or female mice can be used from the PI's existing colony. For one cryopreservation request use no more than 10 females.
It is up to the investigator whether to use 1 female per male or 2 females per male. Both ways work well.
The investigator is responsible for giving the hormones, mating the mice, excising the reproductive tracts and bringing them to the GEM Core R119 by 9 a.m. Any questions should be directed to firstname.lastname@example.org.
Embryo Cryopreservation Instructions
Timing is critical for all steps listed below! Please comply.
1. One week before the scheduled cryopreservation date pick up 1 tube of M2 media, 1 tubes of PMS, and 1 tubes of hCG for each cryopreservation request from Dr. Jianming Xu's lab, N630-Alkek Building. Note: Bring an ice bucket to put the hormones in and put them in a -20 freezer when you get back to your lab. Dilute the hormones right before you go to give the injections.
2. PMS is given at 10 a.m. three days before the scheduled rederivation date (e.g. Tuesday for cryopreservation on Friday). Dilute the PMS with 950 ul of 0.9% saline and administer 0.1cc, i.p. per mouse.
3. h 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.
4. Place the females with the males immediately after administering hCG.
5. The morning following the hCG injections, the females are removed from the male cages.
6. The female mice are cervically dislocated under anesthesia, the reproductive tracts excised, and taken to the Transgenic Core R119. Anesthetize the mouse, make an abdominal incision to expose the intestines, push the intestines upward out of the way to expose the female reproductive tract, excise the oviducts along with the fat pad and ovaries and some of the uterus, no need to isolate only the oviduct.
7. Label the tubes with the request number generated by iLab. Print a copy of the request form and drop it off with the oviducts. Bring the oviducts to the Transgenic Core R119 and leave in the biosafety hood no later than 9 a.m.