cryopreservation is a means of long-term storage of valuable strains.
This technique can be used to safeguard strains of mice or rats from genetic
drift, prevent loss due to disease or catastrophe and reduce animal housing
costs. Embryos are preserved in a cryoprotectant then slowly frozen. Embryos
are then transferred to liquid nitrogen tanks for long-term storage. Cryopreserved
embryos can be recovered at any time the investigator desires. The Transgenic
Animal Facility can cryopreserve embryos from either mice or rats, provide
for their long-term storage in liquid nitrogen and recover strains on
cryopreservation has the following steps:
- Assessment of animal colony
to assure multiple young or proven males are available for matings and
young females are available for superovulation.
- Superovulation of females
to obtain fertilized embryos.
- Cryopreservation of
embryos using a controlled-rate freezer.
- Storage of embryos
in liquid nitrogen tanks.
- Recovery of live animals
from a strain by thawing and surgically transferring the frozen embryos
into pseudopregant recipients.
proceeding with any steps, the investigator needs to meet with the TAF
staff. This consultation will enable us to determine the specific
needs of the investigator so that the proper direction will be taken. We will also provide the investigator with the necessary information
for carrying out the project successfully.
will need to:
- Meet with us to review
your animal colony status. Investigators must have multiple young or
proven breeder males available to obtain fertilized embryos. For mice, TAF recommends having 6-10 proven studs or young males (6-8wks
old). For rats, TAF recommends having 3-6 proven studs or young males
- Determine whether embryos
will be generated from the desired strains by the investigator or by
the TAF staff.
Generation of Embryos for Cryopreservation
animal colony status
procedure for embryo production
request form for service to be provided.
animal transfer form if animals are being housed at the Biotechnology Center
TAF with a copy of latest sentinel serology report
can choose to superovulate/breed their own animals or transfer males to
the quarantine room at the Biotechnology Center for TAF to superovulate/breed.
Investigators choosing to generate their own embryos need to work closely
with the TAF staff to assure success. Investigators choosing to have TAF
generate the embryos, ship male mice or rats from the strain of interest
to the Biotechnology Center. Male mice must come from a pathogen-free
facility and can only be returned to the investigator if direct sentinel
testing is performed (additional charges are billed to investigator for
serology upon completion of project). Stock females are purchased from
spf vendors and used for superovulation.
of where the mice are housed, the procedure to produce the embryos is
generally the same. Young female mice or rats are treated with hormones
to induce superovulation 48 hrs prior to mating. We recommend that
investigators choosing to produce the embryos within their colony follow
our protocol to superovulate mice. For the rat superovulation protocol, click here. Following hormone administration, females are mated one-to-one with males.
Investigators aseptically isolate the reproductive tracts from
the mice on the morning of day 2.5 pc or from rats on the morning of day
3.5 pc and bring them to the TAF staff. This routine will be done
weekly until the desired number of embryos is obtained.
Cryopreservation of Embryos
will collect the embryos and assess their quality based on morphology.
Good quality embryos are preserved in glycerol by slow-cooling methods. Embryos are stored in plastic straws within liquid nitrogen tanks.
recommend freezing 100-125 embryos if homozygous or heterozygous embryos
are preserved. We recommend cryopreserving twice as many embryos
if some of the embryos preserved do not carry the desired modification.
TAF will test viability of each line by thawing one straw of embryos and
transferring them into a pseudopregant recipient. Investigators
have the option of screening offspring and/or incorporating them into
their mouse colony. Additional embryo transfers will be done if
necessary. Fees for this service are charged based on time and material
costs to complete the project.
are stored in TAF. A $75.00 yearly liquid nitrogen storage fee is charged per Principal Investigator in December regardless of the number of samples stored.
can request recovery of strains at any time. Embryos will be thawed
and transferred by TAF. Resulting offspring will be transferred to the
investigator at 3 wks of age, pending serology results and completion
of the animal transfer form by the investigator and the LAR veterinarian from the