Rederivation is accomplished by:
1. Super ovulating 3-5 week old females with hormones.
2. Placing the females with males right after hCG injections.
3. On the scheduled day, euthanize the female mice by cervical dislocation under anesthetic. Collect the reproductive tracts.
4. Collecting the fertilized one cell embryos.
5. Transferring the 1-cell embryos to SPF Day 0.5 pseudo pregnant recipient mice.
6. The pups are born 20 days later and transferred to the investigators animal room when they are 21 days old. We fill out the transfer form and CCM transfers the animals.
7. The investigator weans the pups at 21 days, does tail cuts and screens by PCR or Southern.
The best response to super ovulation is achieved using 3 week old females. Wild type females can be ordered from a vendor or female mice can be used from the PI's existing colony. Both ways work well. For One Colony Expansion slot use no more than 10 females per line.
It is up to the investigator whether to use 1 female per male or 2 females per male. Both ways work well.
The investigator is responsible for giving the hormones, mating the mice, and collecting the oviducts, Steps 1-5. The investigator then places the reproductive tracts in M2 media and brings the oviducts to the GEM Core R119 by 9 a.m. Any question should be directed to firstname.lastname@example.org. The core collects the embryos and transfers them, Steps 4 and 5.
Colony Expansion Instructions
Timing is critical for all steps listed below! Please comply.
1. One week before the scheduled rederivation date pick up 1 tube of M2 media, 1 tube of PMS, and 1 tube of hCG for each rederivation request. The media and hormones are in Dr. Jianming Xu's lab, N630-Alkek Building. Note: Bring an ice bucket to put the hormones in and put them in a -20 freezer when you get back to your lab. Dilute the hormones right before you go to give the injections.
2. PMS is given at 10 a.m. three days before the scheduled rederivation date (e.g. Tuesday for rederivation on Friday). Dilute the PMS with 950 ul of 0.9% saline and administer 0.1cc, i.p. per mouse.
3. hCG is given 47 hours after PMS at 9 a.m., one day before the scheduled rederivation date (e.g. Thursday for rederivation on Friday). Dilute the hCG with 950 ul of 0.9% saline and administer 0.1cc, IP per mouse.
4. Place the females with the males immediately after administering hCG.
5. The morning following the hCG injections, the females are removed from the male cages.
6. The female mice are cervically dislocated under anesthesia, the reproductive tracts excised, and taken to the Transgenic Core R119. Anesthetize the mouse, make an abdominal incision to expose the intestines, push the intestines upward out of the way to expose the female reproductive tract, excise the oviducts along with the fat pad and ovaries and some of the uterus, no need to isolate only the oviduct.
7. Label the tubes with the request number generated by iLab. Print a copy of the request form and drop it off with the oviducts. Bring the oviducts to the Transgenic Core R119 and leave in the biosafety hood no later than 9 a.m.