title: Production of transgenic rabbits using centrifuged pronuclear zygotes.
abstract: Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). 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 zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.
authors.affiliation: YS New Technology Institute Inc, Shimotsuga-Gun, Tochigi, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Hirabayashi
authors.forename: M
authors.initials: M
authors.lastname: Hirao
authors.forename: R
authors.initials: R
authors.lastname: Takahashi
authors.forename: K
authors.initials: K
authors.lastname: Kimura
authors.forename: K
authors.initials: K
authors.lastname: Hirasawa
authors.forename: M
authors.initials: M
authors.lastname: Ueda
authors.forename: S
authors.initials: S
authors.lastname: Hochi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology; D002498:Centrifugation / Q000662:veterinary; D004247:DNA / Q000008:administration & dosage; D005260:Female; D018014:Gene Transfer Techniques / Q000662:veterinary*; D008845:Microinjections / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011817:Rabbits / Q000196:embryology* / Q000235:genetics; D013047:Specific Pathogen-Free Organisms; D013480:Superovulation; D015053:Zygote / Q000254:growth & development*; D017388:Zygote Intrafallopian Transfer / Q000662:veterinary
chemical_list: D004247:DNA