Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes

Adult granulosa donor cells used in the nuclear transfer (NT) procedure can result in cloned cattle. Subsequently, it may be possible to use the same cell type to produce cloned transgenic cattle. Therefore, this study examin ed the effect of genetic manipulation and serum levels in culture of donor granulosa cells on developmental rates and cell number of bovine NT embryos . A primary cell line was established from granulosa cells collected by asp irating ovarian follicles. Cells transfected with a plasmid containing the enhanced green fluorescence protein (EGFP) gene, and non-transfected cells were used for cloning between passage 10 and 15 as serum-starved and serum- fed donor cells. There were no significant differences (P > 0.1) in cleavag e rates or development to the blastocyst stage for NT embryos from transfec ted (60.4 and 13.5%, respectively) or non-transfected (61.9 and 14.1%, resp ectively) and serum-starved (60.6 and 13.4%, respectively) or serum-fed (61 .3 and 14%, respectively) cells. Development rates to blastocyst stage of e mbryos produced using cells at passage 15 (27.1%) were significantly higher than those produced with cells at passage 10, 11, and 13 (7, 11.5, and 14% , respectively, P < 0.05). Green fluorescence was observed at different int ensity levels in all blastocyst stage embryos resulting from transfected do nor cells. The results of the present study indicated that genetically modi fied granulosa cells can be used to produce transgenic NT embryos and prima ry transgenic adult cells at late passage may be more effective donor cells than earlier passaged cells.