Transgene Expression of Green Fluorescent Protein and Germ Line Transmission in Cloned Calves Derived from In Vitro-Transfected Somatic Cells1

Abstract

In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP)-reporter transgene and used as nuclear donor cells in oocyte reconstructions. Because cell synchronization protocols are less effective after transfection, activated oocytes may be more suitable as hosts for nuclear transfer. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either before (metaphase) or after (telophase) activation. Expression of GFP was examined during early embryogenesis, in tissues of cloned calves, and again during embryogenesis, after passage through germ line using semen from the transgenic cloned offspring. Regardless of the kind of host cytoplasm used, GFP became detectable at the 8- to 16-cell stage, approximately 80 h after reconstruction, and remained positive at all later stages. After birth, although cloned calves obtained through both procedures expressed GFP in all tissues examined, expression levels varied both between tissues and between cells within the same tissue, indicating a partial shutdown of GFP expression during cellular differentiation. Moreover, nonexpressing fibroblasts derived from transgenic offspring were unable to direct GFP expression after nuclear transfer and development to the blastocyst stage, suggesting an irreversible silencing of transgenes. Nonetheless, GFP was expressed in approximately half the blastocysts obtained with sperm from a transgenic clone, confirming transmission of the transgene through the germ line.

Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring

“…When the time between activation (by electric stimulation or ionomycin followed by 4-6 h incubation in 6-DMAP or cycloheximide respectively) and NT increased to 4-6 h, development past the eightcell stage was almost completely abolished both with M-or G 0 /G 1 -phase donors (Tani et al 2001(Tani et al , 2003. However, there are cases where artificially pre-activated TII cytoplasts have supported somatic chromatin remodeling after NT in cattle and mice (Bordignon et al 1999(Bordignon et al , 2001, and even resulted in viable cloned goats (Baguisi et al 1999) and cattle (Bordignon & Smith 1998, Kurosaka et al 2002, Bordignon et al 2003. Some of these studies differ from ours in using different species, different activation stimuli (e.g., electrical current, ionomycin, and ethanol/cycloheximide) and activation times, and oocytes of different age post-maturation at the time of telophase enucleation.…”

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

“…Oocytes were matured in vitro as previously described (Bordignon et al 2003). Briefly, cumulus--oocyte complexes (COC) were aspirated from 2 to 7mm follicles and washed in Hepes-buffered TCM199 (Sigma-Aldrich Corporation, St Louis, Missouri, USA) supplemented with 10% (v/v) FBS.…”

Somatic cell nuclear transfer alters peri-implantation trophoblast differentiation in bovine embryos

“…Oocytes obtained from slaughterhouse ovaries were matured in vitro as previously described (Bordignon et al 2003). Briefly, cumulus-oocyte complexes (COC) were aspirated from 2 to 7 mm follicles and washed in HEPES-buffered tissue culture medium (TCM)-199 (Invitrogen Life Technologies) supplemented with 10% fetal bovine serum (FBS).…”