The benefit of mouse line cryopreservation and re-derivation
- Save resources when colonies not in use;
- Limit genetic drift, prevent spontaneous loss of phenotype;
- Protect valuable mouse strain against disasters;
- Rescue mouse line that faces reproductive obstacles;
- Import and export mouse lines.
- Log-in iLab, choose specific service and initiate request.
- Fill in request form, describe project, and indicate the mice location including building name, room, rack and cage card numbers. List how many mice (indicating male or female) you will provide, and their age.
- Submit request.
- Mouse sperm cryopreservation is suited for single mutation and inbred genomic background mouse line. The service will provide cryopreserved sperms in 10 straws. Only two fertile males are required. Recovery of mouse line from cryopreserved sperm is done by in vitro fertilization (IVF). The oocytes donor will be a purchased wild type mouse line; therefore, the recovered mouse line is heterozygous.
- Mouse embryo cryopreservation is suited for multiple mutations and strains with unique genetic background. The recovered mouse line will remain the same genotype. It requires at lease 7 fertile males and 14 females (4-6 weeks) for 5 straws (20 embryos per straw).
Cryopreserve and recovery mouse lines services
Mouse Sperm cryopreservartion
- For each line we need two proven breeder male mice that are at 10 weeks to 10 months old. The males will be on mating about one week and then single housed for 3-5 days prior to sperm collection.
- On the experiment day morning before 9:30am, user will bring the females to an indicated near-by procedure room label the cage with “for TKTC”, and strain name same as on request form.
- We will cryopreserve 10 straws sperms from two healthy males of the line you requested. We suggest to store half number sperm straws in our liquid nitrogen freezer, half number in your own lab. When you need to use the lines again, we will perform in vitro fertilization (IVF) to recover the mouse line.
Mouse embryo cryopreservation
- Stud Male: 4-8 proven breeders, 8 to 20-week old, housed in single cages.
- Female: 8-16 females, 4 to 5 weeks old (adjust upon mouse strain).
- Female superovulation and mating: User will get hormones from TKTC follow Superovulation Instructions to perform superovulation and mating. On the experiment day morning before 9:30am, user will bring the females to an indicated near-by procedure room, label the cage with “for TKTC”. TKTC staff will harvest embryos and proceed cryopreservation.
- We will cryopreserve 100 embryos per line in 5 straws, or repeat the procedure 3 times at maximum if embryo yield is low for some special mouse lines.
- It is important to continue breeding your mice to obtain additional females in the event that the first attempt yields low # of embryos. This will allow the Transgenic Facility to quickly repeat the experiment. Having sufficient females of the correct age is the limiting step in obtaining embryos.
Mouse line re-derivation by fresh embryos
- All mice that are brought into the SIM1 barrier facility need to be re-derived, except for mice that are directly shipped from the production barrier of Stanford VSC-approved vendors including Charles River Laboratories, Harlan, Taconic, and Jax.
- Mouse shipping to non-barrier facilities can also consider fresh embryo re-derivation to reduce quarantine time.
- Re-derived mouse from external line will be housed in Sim1 G0809, and tested with a panel of pathogens. Please reference Timeline and Guidline for users of room Sim1 G0809.
- Provide 3-6 males and 6-12 females per line for heterozygotes; 2-3 males and 4-6 females per line for homozygotes. You can order commercially available females for mating. The mice will be kept in your mouse colony. If your colony is off-campus other arrangements will be made with the VSC.
- Males should be 8-20 week old proven breeders, housed individually. Males will be mated with a test female (e.g., a young sexually mature ICR or CD-1 female) 5 – 7 days before the scheduled re-derivation. This will establish the males as studs and increase the number of zygotes for the re-derivation.
- Females will be superovulated and mated with stud males by yourselves, we will provide superovulation guideline and hormones.
- It is very important to continue breeding your mice in the event that the first rederivation attempt fails, we can quickly repeat the re-derivation.
- You will label the cages with cards indicating "For TKTC". We will arrange a near-by procedure room to harvest embryos, you will bring the cages to the procedure room on rederivation morning. In case you cannot bring the mice to procedure room on rederevation day, please make us accessible to your animal room.
- Surrogate mothers and re-derived mice will be housed in Sim1 G809, please generate a cage sticker sheet for this room. It is strongly recommended that you genotype the pups, even if they are expected to be homozygotes, to ensure you have re-derived strain.
Mouse line re-derivation by in vitro fertilization (IVF)
- We perform IVF using cryopreserved or fresh sperms. The cryopreserved sperm can be from user’s own storage or importing from external institutes.
- We also help to rescue mouse lines that failed natural mating due to aging or illness, please consult with us with details.
- Foster mothers that were implanted embryos by IVF will be nursed in Sim1 G0809, and tested with a panel of pathogens. Please reference Timeline and Guidline for users of room Sim1 G0809. If IVF used sperm from Sim1 barrier facility or venders (Jax, Charles River Laboratories, Harlan and Taconic), foster mother will be nurse in our animal room, no further pathogen test is needed.
Other mouse line services
- Pseudo pregnant mother. We provide 0.5dpc CD1 pseudo pregnant mother. Please request at least two weeks ahead.
- Timed pregnancy. We provide timed pregnant mother at 0.5 day pregnancy for B6, albino B6 and CD1. Please request at least two weeks ahead.
- Cryostorage of sperm or embryos. We provide cryostorage mouse sperm and embryos. One storage unit includes one mouse line or 10 straws or 5 vials. Fee is charged annually (link) at end of calendar year.