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 (memsmail@bcm.tmc.edu).
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.
