Cell donor influences success of producing cattle by somatic cell nuclear transfer.
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% CO(2) and air and some were transferred to MEM, with 5% or 20% O(2) 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.
Biotechnology and Germplasm Laboratory, Agricultural Research Service, USDA, Beltsville, Maryland 20705, USA.
A M
AM
Powell
N C
NC
Talbot
K D
KD
Wells
D E
DE
Kerr
V G
VG
Pursel
R J
RJ
Wall
D000818:Animals; D000831:Animals, Newborn; D002417:Cattle / Q000196:embryology; D002459:Cell Fusion; D019976:Cloning, Organism / Q000379:methods*; D003470:Culture Media; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D008168:Lung / Q000196:embryology; D018482:Muscle, Skeletal / Q000196:embryology; D053652:Nuclear Transfer Techniques; D009865:Oocytes; D010100:Oxygen / Q000378:metabolism; D011247:Pregnancy; D018873:Pregnancy Rate; D014019:Tissue Donors
D003470:Culture Media; D010100:Oxygen