Gene Transfer Methods Pronuclear Microinjection

The primary method used to produce transgenic farm mammals has been the direct microinjection of the transgene into the pronucleus of a zygote (recently fertilized ovum or egg). As in the mouse, pronuclei of rabbit, sheep, and goat zygotes can be readily seen using phase-contrast microscopy or differential interference contrast (DIC) microscopy. Lipid granules in the cytoplasm interfere with visualization of pronuclei in pig and cow zygotes. Centrifugation of pig and cow zygotes can be used to stratify the cytoplasm so that pronuclei are visible with the use of DIC microscopy.[3]

To permit microinjection, ova are placed on a depression slide in a microdrop of media that is overlaid with silicone or paraffin oil to prevent evaporation. The microscope must be equipped with two micromanipulators, one for an egg-holding pipette and the other for an injection pipette. The holding pipette and injection pipette are each fitted with a tube leading to a syringe that permits either gentle suction or carefully controlled fluid injection. As an ovum is held with light suction by the holding pipette, the tip of the injection pipette is inserted through the zona pellucida and cytoplasm into the most visible pronucleus. Several hundred copies of the gene are expelled into the pronucleus. The person performing the injection carefully observes the pronucleus and withdraws the pipette when the pronuclear structure has visibly enlarged. After microinjection, cow embryos are usually cultured in vitro until they are morulae or blastocysts before nonsurgical transfer into the uterus of a synchronous host cow. The injected zygotes of other species are usually cultured only a few hours before they are surgically transferred directly into the oviduct of synchronous host females.

The mechanism by which a transgene integrates into a chromosome is unknown. A transgene usually integrates in a single site on a chromosome, but multiple integrations can occur. Frequently, multiple copies of the transgene integrate in head-to-tail array. Breeding studies with transgenic pigs and sheep indicate mosaicism is a definite problem, with about 20% of founder transgenics failing to transmit the transgene to progeny and another 20 30% transmitting the transgene to less than 50% of their progeny, presumably due to mosaicism in the germ cells as a consequence of integration occurring a few cleavage cycles after microinjection.

The efficiency is usually lower for integration of transgenes into farm animals than into mice. The percentage of gene-injected zygotes that develop into transgenic animals varies from 0.3^1.0% for pigs, 0.1-4.4% for sheep, 1.0-1.7% for goats, and 0.3-2.6% for cattle. A few transgenic chickens have been produced by microinjection of genes into the germinal disk of the recently fertilized egg.[4] After microinjection, the chick embryos were cultured in a host eggshell until hatching time.

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