Protocols

Embryo cryopreservation 

Standard procedure

  • Day 1 - Inject 100 μl (5 u.) of PMS (NHPP) i.p. to the egg donors (1 – 2 females per stud male) at 2:00 (can be 2:00 – 4:00) p.m.
  • Day 3 - Inject 100 μl (3 u.) of hCG (CalBioChem) i.p. to the egg donors at 12:00 (can be 12:00 – 2:00) p.m. Set up mating (1-2 females per stud male).  Please allow 44 – 48 hours between the injections of each hormone.
  • Day 4 - Check female mice for existence of the copulation plug in early morning (before 10:00 a.m.). Please mark base of the tail of the plugged females with a thick black marker pen.
  • Day 5 - Sacrifice all female mice in the morning and collect 2-cell embryos. These eggs will be treated in freezing media and frozen in LN2 immediately after isolation.

 

Number of mice needed for successful freezing and recovering

  • 2 – 3 stud males (or more) per line (preferably 8 – 25 weeks of age, but any age is acceptable if the animals have good breeding history). You keep these mice in your mouse room. We may have to repeat breeding and freezing if available number of stud males or 2-cell embryos harvested is limited.
  • 1 – 2 egg donors (hormone injected females) per stud male per breeding. The core needs at least 4 – 20 females, at 3 to 4 weeks of age, for efficient recovery of fertilized eggs. Please be aware that older females don’t respond to hormones very well, resulting in poor recovery of fertilized eggs. The core can provide necessary number of egg donors if the investigator can use commercially available strain of mice (wild type egg donors).

In order to keep the line homozygous, the investigator needs to provide both egg donors and male breeders. Please note that all females submitted to the core will be sacrificed for collection of fertilized eggs. Enough breeders need to be provided by the investigator beforehand.

 

Materials provided by the core

  • Hormones for induction of superovulation (PMS and hCG); please do not refreeze hCG.
  • Up to 20 egg donors.
  • Embryo culture media, other reagents and supplies.

 

Other reminders

The core can assist the investigator with the hormone injections, confirmation of copulation plug or other procedures if the investigator does not have enough experience in these procedures. Core technicians need to be added to the investigator's animal protocol for this purpose.

Alternatively, the core can perform in vitro fertilization if the investigator’s stock male mice have poor reproductive activity. Please note that the sperm donor(s) will be sacrificed for sperm collection, though special arrangement can be made to save the sperm donor(s). Efficiency of IVF depends on activity and quality of sperm from the donor(s).
The core can provide only wild type egg donors (or sperm donors) for breeding or IVF. Therefore, resulting fertilized eggs for freezing become heterozygous if homozygous animals are provided by the investigator, or segregate into half heterozygous and half wild type if heterozygous breeders are provided.

The investigator may keep part of the frozen embryos in his/her own LN2 freezer for backup.

Egg donors or sperm donors provided by the investigator and submitted to the core need to be transferred to the core’s umbrella protocol (IACUC 003380) before hormone injections.

Please contact the core to set up a meeting for discussing details of the procedure.

 

Rederivation

Outline of rederivation process - How it works

Rederivation by embryo transfer following IVF (in vitro fertilization) is the most effective way to eradicate common pathogens (external or internal parasites, bacterial or viral pathogens such as fur mites, pinworms, helicobacter, MHV, MPV, MVM, etc.) from an infected mouse line. Fertilized eggs or sperm isolated from an infected mouse still carry some of the viral pathogens, which are diluted out by extensive washing and overnight culture in a dish; the intact immune system of a recipient of transferred embryos further contributes to the eradication of the pathogens.

 

Standard procedure

Rederivation procedure requires transferring fertilized eggs produced by infected mice to healthy and clean recipients. Usually, healthy and clean wild type breeders (non-transgenic males or females) are used for producing fertilized eggs in combination with infected males or females. Fertilized eggs produced by breeding infected males and females can still be used for embryo transfer if necessary.

Fertilized eggs are isolated from egg donors after regular breeding, or unfertilized eggs are isolated typically from a large number (usually 10-20) of wild type females purchased from a vendor and fertilized in vitro. You don't have to provide too many infected breeders (mutant mice) if IVF is involved. One sperm donor older than 8 weeks and up to 1.5 years is sufficient unless the donor is weak or in poor condition. You need to provide 5-10, or more, egg donors at 3 to 4.5 weeks of age (or 8-12 with less efficiency) for production of fertilized eggs if you cannot utilize wild type egg donors from a vendor such as JAX or Charles River.

You will have pups about 19 days after embryo transfer, and receive weaned pups 21 days after birth. Starting with 100 fertilized eggs for embryo transfer, about 30 pups would be expected to be produced if all surrogates successfully carry the embryos to term and nurse to weaning.

 

Superovulation

In order to utilize egg donors efficiently, superovulation is induced by injecting them with hormones. They respond well to hormones somewhere between 3-4.5 weeks or 8-12 weeks of age. Inbred strains respond poorly to hormone treatment.

 

Sperm cryopreservation option

Leftover sperm can be cryopreserved after IVF with a nominal fee. Usually, 1-2 males between the ages of 8 weeks to 1.5 years are enough to cryopreserve 10-20 cryotubes of sperm. Successful IVF and cryorecovery is heavily dependent on the activity and quality of the sperm. Long-term storage fee is included in the cost. Ivermectin treatment is not recommended for sperm donors.

 

Summary

Virtually 100% of specific pathogens can be eradicated with IVF followed by embryo transfer.
You may provide egg donors (5-10 or more donors at 3-4.5 or 8-12 weeks of age).
Using wild type egg donors purchased from a vendor is an efficient option and recommended.
We need at least one healthy sperm donor for IVF. Keeping 2-3 more backup mice is helpful.
Tissue samples will be made available for PCR genotyping 7 days after birth.
Rederived pups will be kept under the core's care until weaning, then released to clean vivarium.

 

Microinjection

DNA preparation

Please submit 5 ~10 µg of your construct after digestion(s) followed by inactivation. Run a mini-gel with 10, 20 and 40 ng of sample with markers and attach the picture. The core will isolate the insert from the vector by running an LGT gel, purify and adjust the concentration for microinjection. A control construct may be injected for free or with a nominal extra charge. BAC construct requires a special purification process with a nominal extra charge.

 

ES cell preparation

Please submit at least 1 million ES cells in log phase suspended in ~1.0 ml ES culture medium on ice on the morning of injection. You may provide 2-3 ES clones from the same construct to maximize the chances of germline contribution. Karyotyping and mycoplasma-free status need to be confirmed by the investigator, or by the core with nominal charges, before injection.

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