METHODS FOR PRODUCING TRANSGENIC BOVINE EMBRYOS FROM IN-VITRO MATUREDAND FERTILIZED OOCYTES

Microinjection and in vitro culture procedures were developed to produ ce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM- 199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embry os in vitro for 10 d. In Experiment II, microinjected embryos were cul tured 1) in rabbit oviducts, 2) in vitro in a 5% CO2 in air, or 3) in a 5% CO2 / 5% O2 / 90% N2 incubator. There were no significant differe nces between the 2 in vitro culture environments. The in vitro culture systems supported development-of embryos significantly better than th e rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitr o vs 10% in vivo; 98% of transferred ova were recovered from the rabbi t oviducts. From both experiments, 6 of 92 blastocysts were positive f or the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis.