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Rederivation approaches

Rederivation approaches. April 10, 2012. Goals of this Meeting. Review rederivation options for mouse colonies affected by the recent EDIM outbreak. Sperm cryo followed by IVF and embryo transfer Embryo retrieval and transfer Pros and Cons of the two approaches. Costs.

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Rederivation approaches

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  1. Rederivation approaches April 10, 2012

  2. Goals of this Meeting • Review rederivation options for mouse colonies affected by the recent EDIM outbreak. • Sperm cryo followed by IVF and embryo transfer • Embryo retrieval and transfer • Pros and Cons of the two approaches. • Costs. • Responsibilities of lab and the core No need to take notes, this will be on our web site: http://www.vanderbiltresearch.org/tmescsr

  3. Sperm Cryo followed by IVF & Embryo Transfer Pros: Need only 2 males from the colony—for MOST strains. (Proven breeders younger than 6 months) Very reliable and plentiful frozen stock. Long term safe storage solution. Cons: Resulting offspring are heterozygous (not good for compound mutants). More expensive than embryo rederivation. Recurring fee for storage ($2/straw/year). Cost: Sperm Cryo: 1500 IVF 2500 + cost of mice (varies w strain) Embryo Transfer 1250 5250 total

  4. Sperm Cryo, IVF & Embryo Transfer Responsibility of PI lab: Provide two males, proven breeders 2-6 months of age. Must be able to genotype mice after rederivation. Responsibility of Core: Perform Sperm extraction, Cryo, IVF and Embryo Transfer. Tail resulting pups at 3 weeks of age. Provide long term safe storage for sperm (two separate tanks, two locations).

  5. Sperm Cryo, IVF, Embryo Transfer Timeline: 1. PI submits appropriate service form to TMESCSR. 2. Service forms are reviewed and approved by the Co-Directors of the facility. 3. Experiments are scheduled. 4. PI submits transfer request through DAC for males to be moved on cryopreservation date to Transgenic Mouse/ES Cell Shared Resource procedure room. 5. Cryopreservation is performed and 25 straws per male are placed into long term storage. 6. A small scale IVF is scheduled. 7. One to two straws of sperm is utilized for small scale trial IVF. 8. Confirmation of fertilized embryos at the 2-cell stage. 9. PI notified about results of trial IVF. 10. Pups born, serology, etc. Total time: 3-6 weeks from form submission to IVF, 10 weeks of mouse time.

  6. Embryo Retrieval and Transfer Pros: Can be performed with males and females of a given genotype, therefore better for complex genotypes. No need for IVF (cheaper). Cons: Lab must provide 2-8 males, 2-6 months old, proven breeders and (if necessary) females at age suitable for superovulation (3-4 weeks of age optimal or 7-8 weeks of age). Not a long term storage solution unless the PI opts for Cryo. Cost: Embryo Retrieval and Transfer: 1900 per line + cost of mice (if necessary)

  7. Embryo Retrieval and Transfer Responsibility of PI lab: Provide 2-8 males, proven breeders 2-6 months of age. If necessary, provide 2-8 females of given genotype 3-4 weeks of age, suitable for superovulation. Must be able to genotype mice after rederivation. Responsibility of Core: Prepare foster dams. Isolate and wash embryos. Transfer viable embryos to recipient foster dams. Tail resulting pups at 3 weeks of age.

  8. Embryo Retrieval and Transfer Timeline: 1. PI submits appropriate service form to TMESCSR. 2. Service forms are reviewed and approved by the Co-Directors of the facility. Experiments are scheduled and females ordered (if necessary). Females are superovulated (in TMESCSR space if wild type and in PI space if necessary). Mice are mated. Embryos are retrieved and transferred into pseudo-pregnant foster dams. Resulting pups are tailed at 3 weeks. Total time: 3-4 weeks from form submission to mating and embryo retrieval/transfer, 10 weeks of mouse time. (Note, this will vary if the lab chooses not to use wild type females).

  9. What happens next? • Mice are born in the barrier (TMESCSR space). • Mice are tailed and earmarked and tail snips provided to you for genotyping. • Genotype results need to be returned to the core by the time pups are 6 weeks of age. • Dams are bled for serology at weaning. At 6 weeks of age, 2 pups from each litter are bled for serology. • Serology results return and are reviewed by DAC. • Clean mice of desired genotype are exported.

  10. Next steps Choose which approach you wish to take (sperm vs embryo). Fill out paperwork. We will schedule experiments. Can email Jennifer or me to discuss this further. Things to note: complex breeding strategies, numbers and ages of males and females available, strain background. jennifer.skelton@vanderbilt.edu trish.labosky@vanderbilt.edu http://www.vanderbiltresearch.org/tmescsr

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