title: Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy.
abstract: Oocytes collected by laparoscopic ovum pick-up (LOPU) were successfully used to produce transgenic goats by pronuclear microinjection of in vitro zygotes. Estrus cycles of 109 donor goats were synchronized using intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate and treatment with 70 mg NIH-FSH-P1 and 300 IU eCG to stimulate follicular development. Follicles were aspirated under laparoscopic observation. In vitro maturation (IVM) of oocytes was performed in M199 supplemented with hormones, kanamycin and 10% estrus goat serum. Following IVM, oocytes were cocultured with capacitated semen in TALP supplemented with 20% estrus goat serum for 15-20 h. The resulting zygotes were microinjected with a linear DNA fragment. In total, 3293 follicles were aspirated (15.7+/-9 follicles aspirated per donor) and 2823 oocytes were recovered (13.4+/-8 oocytes per donor). A total of 1366 zygotes were microinjected and transferred into 219 recipient goats by midventral laparotomy (average 6.2 embryos per recipient). A total of 150 kids were born, of which 9 (6 M: 3 F) were confirmed to be transgenic by PCR and Southern blotting analyses. These results demonstrate that acceptable transgenesis rates can be obtained in goats by DNA microinjection of in vitro produced zygotes.
authors.affiliation: Nexia Biotechnologies Inc., 320 St. Georges, Que., St. Telesphore, Canada J0P 1G0. hbaldassarre@nexiabiotech.com
authors.forename: Hernan
authors.initials: H
authors.lastname: Baldassarre
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Nacereddine
authors.initials: N
authors.lastname: Kafidi
authors.forename: Melanie
authors.initials: M
authors.lastname: Gauthier
authors.forename: Nathalie
authors.initials: N
authors.lastname: Neveu
authors.forename: Jamie
authors.initials: J
authors.lastname: Lapointe
authors.forename: Laura
authors.initials: L
authors.lastname: Sneek
authors.forename: Martin
authors.initials: M
authors.lastname: Leduc
authors.forename: F
authors.initials: F
authors.lastname: Duguay
authors.forename: J F
authors.initials: JF
authors.lastname: Zhou
authors.forename: A
authors.initials: A
authors.lastname: Lazaris
authors.forename: C N
authors.initials: CN
authors.lastname: Karatzas
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000502:physiology; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D006041:Goats / Q000235:genetics* / Q000502:physiology; D010535:Laparoscopy / Q000662:veterinary; D008845:Microinjections / Q000662:veterinary; D009865:Oocytes / Q000502:physiology*; D006080:Ovarian Follicle / Q000166:cytology; D015053:Zygote / Q000502:physiology*
chemical_list: D004247:DNA