1. Immature donor females are superovulated and mated with stud males (Subheading 5.).
2. Approximately 12 h postcoitus (pc), the oviducts from donor females are collected, and eggs are harvested and placed into culture (Subheading 8.).
3. The eggs are microinjected with purified cloned DNA (Subheading 11.).
4. Eggs that survive microinjection are returned to the natural environment by implantation into pseudopregnant surrogate mothers produced by mating sexually mature females with vasectomized males (Subheading 13.).
5. A percentage of the transplanted eggs will survive to full term, and will be delivered either naturally or by Caesarean section (Subheading 14.).
6. Transgenic animals are identified by analysis of genomic DNA isolate from the tail tissue.
7. Transgenic animals are bred to produce a line and analyzed for transgene expression.
The efficiency of producing transgenic mice or rats varies considerably between experiments. Mice have been more often used in transgenic experiments, and the efficiencies of each stage are well recorded. Under optimal conditions, 60-80% of the eggs survive injection, and 10-30% implant in the pseudopregnant recipient, proceed through normal development, and are born. Ten to 30% of the pups are transgenic. In rats, the available data are that 50-75% survive injection, 10-30% implant, and are born as pups of which 10-33% are transgenic (8).
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