Cell Donor Influences Success of Producing Cattle by Somatic Cell Nuclear Transfer

• Published in: Biology of reproduction 2004-07, Vol.71 (1), p.210-216
• Main Authors: Powell, A. M, Talbot, N. C, Wells, K. D, Kerr, D. E, Pursel, V. G, Wall, R. J
• Format: Article
• Language: English
• Subjects: Animals; Animals, Newborn; Biological and medical sciences; blastocyst; cattle; Cattle - embryology; Cell Fusion; Cloning, Organism - methods; Contents; Culture Media; early development; embryo; embryo culture; Embryo Transfer; Embryo, Mammalian - cytology; embryogenesis; Embryology: invertebrates and vertebrates. Teratology; Female; fetus; fibroblasts; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Lung - embryology; Molecular embryology; Muscle, Skeletal - embryology; nuclear donor cells; Nuclear Transfer Techniques; nuclear transplantation; Oocytes; oxygen; Oxygen - metabolism; oxygen concentration; oxygen tension; Pregnancy; pregnancy outcome; Pregnancy Rate; serum; somatic cells; Tissue Donors; Vertebrates: reproduction
• ISSN: 0006-3363; 1529-7268; 1529-7268
• DOI: 10.1095/biolreprod.104.027193

To assess sources of variation in nuclear transfer efficiency, bovine fetal fibroblasts (BFF), harvested from six Jersey fetuses, were cultured under various conditions. After transfection, frozen-thawed lung or muscle BFF donor cells were initially cultured in DMEM in 5% CO2 and air and some were transferred to MEM, with 5% or 20% O2 or 0.5% or 10% serum and G418 for 2–3 wk. Selected clonal transfected fibroblasts were fused to enucleated oocytes. Fused couplets (n = 4007), activated with ionomycin and 6-dimethylaminopurine, yielded 927 blastocysts, and 650 were transferred to 330 recipients. Fusion rate was influenced by oxygen tension in a fetus-dependent manner (P < 0.001). Blastocyst development was influenced in a number of ways. Hip fibroblast generated more blastocysts when cultured in MEM (P < 0.001). The influence of serum concentration was fetus dependent (P < 0.001) and exposing fibroblast to low oxygen was detrimental to blastocyst development (P < 0.001). Cells from two of the six fetuses produced embryos that maintained pregnancies to term, resulting in eight viable calves. Pregnancy rates 56 days after transfer for the two productive donor fetuses, was at least double that of other recipients and may provide a fitness indicator of BFF cell sources for nuclear transfer. We conclude that a significant component in determining somatic cell nuclear transfer success is the source of the nuclear donor cells.