Below is the protocol for the isolation of the reproductive tracks (collection of oviducts)
for Strain Rederivation and Cryopreservation. Care should be taken in following the protocol
to ensure that Specific Pathogen Free embryos can be collected for rederivation and/or
cryopreservation. If you have any questions about this procedure please contact memsmail@bcm.tmc.edu
and arrangements will be made for someone in Dr. DeMayo's lab to show you how harvest the
reproductive tracts.
The goal of rederivation of mouse strains is to achieve strains of mice that are pathogen free.
The goal of cryopreservation is to store pathogen free strains. The GEM Core exploits the
manipulation of the one cell mouse embryo as a means of rapidly and efficiently collecting
SPF mouse embryos. The procedure below assumes that you are collecting reproductive tracts
from non SPF mice in a non-SPF facility. However, if you are collecting embryos in a SPF
facility the same precautions should be taken.
Rederivation/Embryo Freezing Protocol-Mice from non-SPF Facility
Where?
Put your females in clean cages and bring them from
the non-SPF facility to a procedure space or to your lab space. (If the animals are in a SPF
facility the procedure is done in a procedure room). When you have finished dispose of the
carcasses immediately.
When?
Begin the collection at 8:00am (or earlier) on the day after
you have given the hCG and mated your females. Complete the collection and hand the oviducts off
to another person in your lab to bring to the Transgenic Core in TMF, room V016 by 9:00.
If you cannot hand off the reproductive tracts to another person or do not have access to
TMF, deliver the reproductive tracts to Dr. DeMayo's lab (M725 Debakey). If you are delivering
to the DeMayo Lab make sure the GEM Core is notified ahead of time that you are doing so.
What to have on hand when you collect:
1 clean diaper for each line of mice
2 pair of gloves for each line you are collecting from
1 squirt bottle of 70% alcohol
1 set of tools for collecting the oviducts
1 extra clean pair of forceps (VERY IMPORTANT!! Wipe with alcohol after each set of collections!)
leave alone 1 tube with two mls of M2 media for each line you are rederiving/freezing
(pick the media up from Dr. DeMayo's lab, M725-DeBakey and divide into 2 tubes.)
Sharpie for labeling the second tube. Please write legibly. (KEEP THE SHARPIE CLEAN!)
Procedure:
1.
Bring supplies to your work area. Wipe everything down with
alcohol. Lay out your diaper.
2.
Bring your animals to your work area. Collect from one line
at a time. All females from the same line should be collected
at the same time.
3.
Put on a new pair of gloves. Euthanize the female mice,thoroughly
wash them with alcohol.
4.
Cut out each oviduct and place them in the first tube. Collecting
the "oviducts" means removing the reproductive tract
out 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. When all oviducts for the first line have been collected,
roll your diaper with the females up and put it to the side.
5.
Take off your gloves, put on a clean pair of gloves. Pick
up your clean forceps and transfer the oviducts from the first
tube of M2 to the second tube of M2. Cap the tube. Clean the
outside with alcohol, dry the tube, then label it. Set this
tube aside. Wipe the pair of forceps with alcohol.
6.
Lay out a new diaper for the second line and continue as before
until all of your lines have been collected.
7.
Clean the work surface with alcohol.
8.
Hand the oviducts off to another person to bring to the Transgenic
Core in TMF, room V016. If you cannot hand off the reproductive
tracts to another person or do not have access to TMF, deliver
the reproductive tracts to Dr. DeMayo's lab (M725 Debakey).
If you are delivering to the DeMayo Lab M725 make sure that
the GEM Core is notified ahead of time that you are doing so. Deliver the
oviducts by 9:00 a.m.
