Alternate Services for Cryopreservation and Re-derivation of Mouse Lines
1. Sperm Cryopreservation. For sperm cryopreservation at least 3-5 males between the ages of 8 weeks and 6 months are optimal. For animals that cannot be transferred to the CRB facility, the isolation must be done by the user in his/her own facility. This requires hands-on training of lab personnel for the epididymis harvest. Media will be supplied by the TCMF to all labs. Five straws of sperm per male will be frozen. For this service, we recommend a one-time harvest of embryos to be frozen prior to sacrificing the males for sperm cryopreservation. Cryopreserved samples can be stored and maintained by the Core at the fee of $24.00/line/year.
2. Embryo Cryopreservation. For animals that cannot be transferred to the CRB facility, the mating to generate embryos and the subsequent isolation of the oviducts must be carried out by the user in his/her own facility. Specifically, this requires hands-on training of lab personnel in intraperitoneal (ip) hormone injection and oviduct harvest. All media and hormones will be supplied by the facility as will the actual training. The user’s lab will be responsible for two ip hormone injections, 48 hrs apart between the hours of 12 PM and 2 PM, into a set of 3-week old females. Once the second hormone injection is complete the female mice should be housed with single caged males and left alone for 2.5 days. On the morning of harvest, the females will be sacrificed by the user, oviducts removed and a lab member who has not been near the actual animals must deliver the organs to the TCMF (behind the microbiological barrier) in doubled Petri dishes. Approximately 150 embryos from a homozygous line and 300 embryos from a heterozygous line will be frozen. Once cryopreservation is complete, a quality control test is done once by removing one straw and determining the rate at which the frozen embryos develop into blastocysts. A minimum of 5-7 males at the age of 2-6 months along with 1-2 females per male for each round. Various rounds are usually required to complete the cryopreservation of a line. Cryopreserved samples can be stored and maintained by the Core at the fee of $24.00/line/year.
3. Re-derivation by IVF. The epididymis of the donor male will be harvested by the user (as in 1., above), delivered to the TCMF, and the released sperm will be used for IVF of wild-type oocytes. The oocytes will have to be harvested in the TCMF from females acquired from approved vendors. Fertilized oocytes will be cultured overnight and any resulting 2-cell stage embryos will be transferred into surrogate females. The transplanted pseudo-pregnant recipients will then be then transferred to the John Morgan building for gestation and quarantine monitoring. ULAR diagnostics must be contacted and space within the quarantine cleared before any date for IVF can be set. The user should consult with ULAR regarding duration and rules of quarantine in John Morgan.
4. Re-derivation by Embryo Transfer. This requires hands-on training of lab personnel to perform intraperitoneal (ip) hormone injection and to dissect uterine horns. All media and hormones will be supplied by the facility as will the actual training. The user’s lab will be responsible for two ip hormone injections, 48 hrs apart between the hours of 12 PM and 2 PM, into a set of 3-week old females. Once the second hormone injection is complete the female mice should be housed with single-caged males and left alone for 3.5 days. On the morning of harvest, the females will be sacrificed by the user, uterine horns removed and a lab member who has not been near the actual animals must deliver the organs to the TCMF (behind the microbiological barrier) in doubled Petri dishes. ULAR diagnostics must be contacted and space within the John Morgan quarantine cleared before any date for embryo harvest can be set. The user should consult with ULAR regarding the duration and rules of quarantine in John Morgan.