Generation of dwarf coat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes

The developmental potential of caprine fetal fibroblast nuclei after in vit ro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Ce lls were transfected with constructs containing the enhanced green fluoresc ent protein (eGFP) and neomycin resistance genes and were selected with G41 8. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% f etal calf serum for 4-8 days prior to use in NT. Immature oocytes were reco vered by laparoscopic ovum pick-up and matured for 24 h prior to enucleatio n and NT. Reconstructed embryos were transferred as cleaved embryos into sy nchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 re cipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultras ound. Of these, four recipients delivered five male kids (7.1% of embryos t ransferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase cha in reaction, Southern blotting, and fluorescent in situ hybridization analy ses. Nuclear transfer derivation from the donor cells was confirmed by sing le-strand confirmation polymorphism analysis. These results demonstrate tha t both in vitro-transfected and nontransfected caprine fetal fibroblasts ca n direct full-term development following NT.