Embryo Cryopreservation

•  Introduction

Embryo 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 demand.

Embryo cryopreservation has the following steps:

1. Assessment of animal colony to assure multiple young or proven males are available for matings and young females are available for superovulation.
2. Superovulation of females to obtain fertilized embryos.
3. Cryopreservation of embryos using a controlled-rate freezer.
4. Storage of embryos in liquid nitrogen tanks.
5. Recovery of live animals from a strain by thawing and surgically transferring the frozen embryos into pseudopregant recipients.

•  Getting Started

Before 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.

You will need to:

1. 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 (8-10wks old).
2. Determine whether embryos will be generated from the desired strains by the investigator or by the TAF staff.

•  Generation of Embryos for Cryopreservation

Checklist:

1. Review animal colony status
2. Review procedure for embryo production
3. Complete a billing request form for service to be provided.
4. Complete animal transfer form if animals are being housed at the Biotechnology Center
5. Provide TAF with a copy of latest sentinel serology report

Investigators 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.

Regardless 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

TAF 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.

We 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.

•  Long-term storage/recovery

Embryos 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.

Investigators 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 relevant school.