SOP Rederivation - Embryos by In Vitro Fertilization

1. Fill out online requisition form: http://med.stanford.edu/transgenic/form_embryoderivation.html.

2. Confirm rederivation date with Andreea Salinas (nandreea@stanford.edu)

3. Provide 2 males per line. Males must be 8-20 week old and proven breeders. Males will be euthanized for their sperm.

4. Provide 10 females per line at 4 - 5 weeks of age. Females will be superovulated by the Transgenic Facility. The females will then be euthanized and their oocytes harvested and fertilized with the sperm from your males to obtain the embryos for rederivation.

a. If you are able to use commercially available females, inform the Transgenic Facility what strain of females to order (vendor, strain, catalog number).

5. If mice are on campus, contact Charlene Wang (cwang8@stanford.edu) to arrange a meeting with a Transgenic Facility technician.

a. You will be given cage cards to label the male and female cages.

b. Provide the Transgenic Facility with access information to your mouse room so they can hormone prime your mice.

c. Provide a sheet of bar code labels with your account number.

6. If mice are off-campus, allow one week before the scheduled rederivation date.

a. One week prior to the scheduled rederivation date, the mice (males, females) will be transferred to RAF 1, AF077D.

b. Email Andreea (nandreea@stanford.edu) to set-up the transfer of your cages to RAF 1, room AF077D. Andrea will need the following information: Facility where your mice are housed; Room Number; Rack Number, Rack Side, Rack Shelf (shelves listed from top down); Barcode numbers Example: Arastradero, Room B148, Rack 5, Side A, Shelf 3, Barcode numbers: #123222 to 123229 (8 cages).

c. Provide a sheet of bar code labels with your account number.

7. It is strongly recommended that you genotype any rederived pups, even if they are expected to be homozygotes, to ensure you have rederived your strain.

8. If genotyping is necessary, e.g., your rederived pups are a mixture of wild-type and transgenic genotypes, you MUST perform the genotyping quickly to determine if you have obtained your strain. All the rederived pups may be wild-type or you may obtain only females (or males) of the correct genotype necessitating repeating the rederivation to obtain the other sex. The longer you delay, the older your females and stud males will be. If you cannot genotype the pups quickly, the use of a commercial genotyping service, e.g., Transnetyx, is recommended.

9. It is important to continue breeding your mice to obtain an additional 10 - 14 females in the event that the first rederivation fails or results in low# of pups. This will allow the Transgenic Facility to quickly repeat the rederivation. Having sufficient females of the correct age is the limiting step in embryo transfer rederivation.