Pronuclear injections to produce transgenic mice:

1. Egg production for injections - To obtain a large quantity (>250) of eggs for injection, sexually immature FVB/N females (3-5 weeks of age) are superovulated by subcutaneous injecting PMS and HCG hormone 46-48 hours apart.  Females are mated to stud males immediately following the HCG injection.
   
   
2. Harvesting the eggs - Eggs are harvested the next day from the ampulla of the oviduct of the mated females.  Eggs are treated with hyaluronidase to remove nurse cells, and are then washed through several drops of M2 (or any equivalent handling media, e.g. CZB/HEPES).  Fertilized eggs are cultured in M16 media (or any equivalent culturing media, e.g. CZB) at 37°C and in 5.0% CO₂ incubator until injection.
   
   
3. Injecting the eggs - 30-50 eggs are removed from the incubator at a time for injection.  Each egg is individually injected with the DNA fragment of the day under high magnification.  When each egg in that group has been injected, all the survival eggs are returned to the culture medium M16 in the incubator.  This procedure is repeated until all eggs have been injected.
   
   
4. Implanting the eggs - Injected eggs are then surgically transferred in goups of 10-15 bilaterally or 25-35 unilaterally into the oviduct of pseudopregnant females (females which have been mated to vasectomized males).  The animals are allowed to recover from anaestheisa on a warming plate, and returned to the animal room.  They are kept unter sterile conditions throughout their pregnancy.