title: Nuclear transfer in the bovine using microinjected donor embryos: assessment of development and deoxyribonucleic acid detection frequency.
abstract: Bovine embryos that had been microinjected with DNA were examined for their potential use as donor embryos in nuclear transfer. Donor embryos were obtained from oocytes collected by transvaginal oocyte aspiration, matured and fertilized in vitro, microinjected with a murine whey acidic protein-human protein C genomic DNA construct, and cultured in vitro on liver cells of buffalo rat (Rattus norvegicus). Blastomeres from these embryos were transferred into enucleated bovine oocytes received from an abattoir by electrofusion at 40 h postmaturation. Following 7 d of culture, the developmental stage was recorded, and resulting embryos were prepared for analysis by polymerase chain reaction. Embryos that were derived from microinjected donor embryos did not differ from control donor embryos (11 vs. 8.6%) in development to the morula and blastocyst stage. Of the biopsies from 20 microinjected donor embryos, 19 were positive for the injected DNA. Of 37 embryos developing normally, only 12 (32.4%) were positive for the injected DNA. These results indicate that microinjected embryos can be successfully used in a nuclear transfer program to produce additional viable embryos and that these embryos may be reliably screened for the transgene for transfer to recipients.
authors.affiliation: Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Krisher
authors.forename: J R
authors.initials: JR
authors.lastname: Gibbons
authors.forename: F C
authors.initials: FC
authors.lastname: Gwazdauskas
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001755:Blastocyst / Q000502:physiology; D002417:Cattle / Q000196:embryology*; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage / Q000032:analysis; D005307:Fertilization in Vitro; D018014:Gene Transfer Techniques; D008099:Liver; D008845:Microinjections; D008894:Milk Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009028:Morula / Q000502:physiology; D053652:Nuclear Transfer Techniques; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000235:genetics; D051381:Rats
chemical_list: D008894:Milk Proteins; D011486:Protein C; C024471:whey acidic proteins; D004247:DNA