MOUSE SUPEROVULATION AND EMBRYO PRODUCTION
SUPEROVULATION AND EMBRYO PRODUCTION
Note: The following protocol is targeted at those investigators who wish to use females generated in their own colonies. If wild-type, vendor supplied donor females are to be used, the TGTC will order the desired donors and perform the superovulation injections within our colony room or other designated area. Requirements for breeder males from your colony remain as described below.
Three week old females are generally used as embryo donors for most strains. The donors are hormonally primed to induce overproduction of the oocytes (superovulation). The superovulated donors are then mated overnight and embryos are isolated the next morning. The timing of the hormone injections and matings are critical for the successful generation of embryos.
Any deviations from the timing of the hormone injections and mating may result in low embryo production and/or low fertilization. If insufficient numbers of fertile embryos are produced for our use, the procedure will have to be repeated and additional charges to you will apply.
IFYOU ARE USING YOUR OWN EMBRYO DONORS:
ANIMAL REQUIREMENTS:
· Investigator must supply a minimum of six donor females in the specified age range (21-28 days of age at the time of the first hormone injection). Please note that it will take about six – seven weeks to produce the proper aged females.
· Sexually mature breeder males between 8 weeks and six months of age are required. These males must have successfully mated within 1-2 weeks prior to your scheduled cryopreservation date. Successful mating is defined as the ability of the male to plug a female within 1-5 days. The presence of a vaginal plug is indicative of successful mating. If a male has not plugged a female within this time span, do not use him.
· Check for vaginal plugs daily between 8 and 10 AM. Please contact the Transgenic Core if you need assistance in setting up a test mating. It is not necessary to use your valuable females for these tests. Wild type or otherwise unnecessary mature females may be used.
· When the females to be used for donors are about one week old, contact the TGTC so that your procedure can be scheduled.
· You will administer the hormone as scheduled below.
The Transgenic and Gene Targeting Core will provide the hormones required for superovulation. Store any vials of hormone at -20° to -70° C until use. Please arrange for hormone pickup from the TGTC at least 1-2 weeks before your scheduled cryopreservation or rederivation.
IF YOU ARE USING VENDOR-SUPPLIED EMBRYO DONORS:
The TGTC will schedule your procedure and order the required number and strain of embryo donors. Upon arrival, we will begin superovulation of your donor mice.
On the day prior to your scheduled cryopreservation or rederivation, please pick up your superovulated donors at the TGTC (RC2, room 0384) between noon and 1PM for mating with your male mice. The following morning, wrap the cages loosely in a garbage bag, spray the outside with disinfectant and contact the TGTC for instructions on delivering the embryo donors.
IF YOU NEED HELP: If you need assistance with any procedure described in this protocol, please contact the Transgenic Core at . Our staff cannot enter your animal room. However, we can provide you with further instructions and can assist you in obtaining help from trained CCM staff to conduct the injections and matings.
SUPEROVULATION SCHEDULE
Day One: PMSG Injections; 2PM
Inject each donor female intraperitoneally (IP) with 0.1 ml PMSG in saline (provided)
There is no need to single house the donors during the hormone regimen.
Day Two: No procedures scheduled
Day Three: hCG Injections/Mating; 12 Noon
Inject each donor female IP with 0.1 ml of 50 IU/ml hCG in saline (as supplied).
Immediately following hCG injection, mate each donor female with a breeder male. At this stage, house one male with one female
Day Four (early morning, 8-10am): Check whether the females show a vaginal plug (evidence of mating).
Transfer females to the Transgenic and Gene Targeting Core (up to five females can be housed in one cage at this time). Make arrangements with the core staff to pick up your donor females.
NOTES AND GENERAL INFORMATION
· TGTC personnel cannot perform the hormone injections for the investigator when you are using your own donor females and stud males. We are not allowed to enter investigator animal rooms in order to minimize exposure to animal pathogens and to prevent any possibility of contaminating the Transgenic Core animal colonies.
· The injections are straightforward and can be easily performed by your laboratory personnel. If preferred, you can contact the CCM Veterinary Services staff and arrange to have the veterinary technicians perform the injections for you. CCM Veterinary Services would charge a small fee for the service.
· Both hormone solutions (PMSG and hCG) as provided contain 50 IU/1ml in saline. Dosage for each mouse is 0.1ml of either hormone administered intraperitoneally (IP). Let us know how many animals are to be superovulated so that you get sufficient hormone solution.
· The hormones must be kept frozen preferably at -80°C until use. Thaw solutions just prior to use. Once thawed, the hormones degrade and unused solutions should be discarded. Do Not Re-freeze!
· It is strongly advised that 1 cc tuberculin syringes with fixed 28 ga. x ⅜ inch needles be used for injecting the donors. Contact the CCM Veterinary Services staff to obtain these syringes.
It is important for the success of the superovulation experiment that you adhere strictly to procedures outlined in this protocol.
Potential problems that could affect the efficiency of superovulation experiments:
· Females past puberty (over 4 weeks of age) generally respond poorly to the hormonally-induced superovulation, thus producing lower numbers of embryos. Make sure the females you use are between 21-28 days of age!
· Males are too old.
· It is strongly advised that the breeder males be mated to sexually mature females (around 8 weeks of age for most inbred strains) about three weeks prior to use in superovulation. This will stimulate sperm production resulting in greater male fertility and thus increase the percentage of fertilized embryos that we can recover for cryopreservation and rederivation.
If you have further questions regarding colony setup and superovulation, please send an email to us at Makeamouse.
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So_emb_prod.doc v2.5 7/18/10 ja-sw