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Embryo Cryopreservation

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. Collecting the reproductive tracts the morning after placing the female with the male.
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. Both ways work well. For one freezing slot use 10 females per line (no more).
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, collecting the oviducts, Steps 1-5. The investigator then places the reproductive tracts in M2 media and bring them to the GEM Core in TMF, room V016 by 9:00 am. If you do not have access to TMF bring the oviducts to Dr. DeMayo's lab, M725 Debakey Building. Please let us know if you will be dropping the oviducts off in Dr. DeMayo's lab. The Core collects the embryos and freezes them, Steps 6 and 7.

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 tube of PMS, and 1 tube of hCG for each cryopreservation request from Dr. DeMayo's lab, M725-DeBakey 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 am four days before the scheduled cryopreservation date( eg. Monday for cryopreservation on Thursday). Dilute the PMS with 950 ul of 0.9% saline and administer 0.1cc, of PMS IP per mouse.
3. hCG is given 47 hours after PMS at 9 am, one day before the scheduled cryopreservation date (eg. Wednesday for Thursday cryopreservation). 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 euthanized and the oviducts are collected and taken to the Transgenic Core. Collecting the "oviducts" means removing the reproductive tract from the ovarian fat pad to the top of the uterus, leaving a small portion of the uterus attached to ensure that the oviduct is intact. If you have any questions about this procedure please contact MEMS ( Arrangements will be made for someone in Dr. DeMayo's lab to show you how to harvest the reproductive tracts.
7. Label the tubes with the request number generated by MEMS. Print a copy of the request form and drop it off with the oviducts. Bring the oviducts to the Transgenic Core, TMF Room V016, no later than 9:00 or to Dr. DeMayo's lab (M725 Debakey) if you do not have TMF access.