Production of transgenic rabbits using centrifuged pronuclear zygotes

Superovulation of female rabbits was induced by subcutaneous injection(s) o f porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and w ere classified into two categories under a microscope equipped with Nomarsk i interference-contrast optics at x 200 magnification: (A) zygotes with cle arly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 m in cenuifugation at 12,000 x g. No significant difference between strains w as found in the proportion of category-a zygotes (JW 72.6% vs NZW 79.3%). P ronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugat ion toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution ( 5 mug/ml of fusion gene composed of bovine alphaS(1)-casein promoter and hu man growth hormone structural gene) was microinjected into the pronucleus o f the JW zygotes. The pronucleus of category-a zygotes with a mean volume o f 7.4 pi swelled up to 16.6 pi (132% increase), while that of category-B zy gotes with a mean volume of 6.1 pi swelled up to 15.9 pi (148% increase). N evertheless, similar proportions of category-A and category-B zygotes devel oped into offspring after transfer to recipient females (11.1 and 11.2%, re spectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygot es(P>0.05). To date, transgenic rabbits have been produced without centrifu gation of pronuclear zygotes. However approximately 25% of fertilized rabbi t zygotes can be used for DNA microinjection after they have been centrifug ed to visualize their pronuclei.