Superovulation of Mice
Given each mouse 0.05 ml PMSG (Straight from vial), administered i/p at 12 o'clock midday
46 - 48 HOURS LATER (i.e. 10am - 12noon, two days later)
Dilute 0.5ml hCG (in vial) in 2ml PBS
Of this mixture, give each female mouse 0.05ml, administered i/p. Place females with males straight away.
Between 9 am and 11 am the next day, mice should be checked for copulation plugs.
Note: Recipient mice (not superovulated and placed with vasectomized stud males) usually mate on the third night after being placed with the boys. To ensure that sufficient recipients are mated, place recipients-to-be with males three days before the required plugging date.
Human Chonionic Gonadotrophin (hCG)
Pregnant Mares Serum Gonadotrophin (PMSG)
PMSG AND hCG
PMSG and hCG manufactured by:
Chorulon (hCG or chorionic gonadotrophin) comes in concentrations of 500 i.u., 1500 i.u. or 5000 i.u. The one we use is the 5000 i.u. in a vial. Using the 5 ml of solvent provided, the chorulon is dissolved and removed from the vial with a syringe containing 5 ml PBS. This brings the concentration of chorulon to 500 i.u./ml (10 ml soln). It is distributed into 20 eppendorfs (0.5 ml per tube) and frozen. To work with, a 100 i.u./ml conc. is required so it should be diluted 1/5 before use i.e. 0.5 ml chorulon in 2 ml PBS.
Folligon (PMSG or pregnant mouse serum gonadotrophin) comes in 1,000 i.u., 5,000 i.u. and 20,000 i.u. concentrations. We use the 1000 i.u. vial. Using a syringe, the 5 ml of solvent provided is removed from its vial and mixed into the folligon vial. The folligon/solvent mix is then removed from its vial by syringe, already containing 5 ml PBS. Then distributed into 10 eppendorfs (1 ml per tube) and freeze. This gives us a conc. of 100 i.u./ml. This is the required working conc. so dilution is not necessary.
|Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: email@example.com |
David Bowtell PMCI October 1998