Production of transgenic goats by somatic cell nuclear transfer.

摘要

A series of experiments were conducted to produce transgenic goats by somatic cell nuclear transfer (NT). In all experiments, donor cells were electrically fused to enucleated metaphase II oocytes, then chemically activated. In a preliminary study to evaluate embryonic development following NT with proliferating or quiescent fibroblast or cumulus cells, significantly more embryos reconstructed with quiescent cumulus cells fused (77%) compared with proliferating cumulus cells (41%), proliferating fibroblasts (36%) or quiescent fibroblasts (37%). Improved development to the eight- to sixteen-cell stage was observed when fibroblast cells were serum starved (serum starved 39% vs. serum fed 15%). However, there was no benefit of serum starvation for cumulus cells nor was there a difference among the treatments for development to the blastocyst stage. Next, the in vivo developmental potential of NT embryos produced from the fusion of quiescent transgenic donor cells with cytoplasts prepared from either FSH-stimulated ovaries or nonstimulated abattoir-derived ovaries was compared. There was no difference in the number of transferable embryos produced, nor was there a difference in the number of pregnancies established per recipient between either treatment. All pregnancies from both groups culminated in the births of five healthy female kids. In the third and fourth experiments, proliferating and quiescent donor cells from two different transfected fibroblast cell lines were used to generate cloned goats capable of producing human recombinant antibodies in milk. There was no difference in the number of transferable embryos produced from proliferating donor cells compared with quiescent cells, nor was there a difference in the number of pregnancies established per recipient between either treatment. A twin pregnancy from the quiescent treatment resulted in the birth of two healthy transgenic kids. In the final study, oocytes were harvested either from FSH-stimulated ovaries or from nonstimulated abattoir-derived ovaries to generate transgenic goats by NT using fetal fibroblast cells transfected with the MSP-1₄₂ gene. Following transfer of the reconstructed embryos to recipient females, one healthy transgenic kid was produced. There was no effect of oocyte source on the number of pregnancies established or on the number of offspring produced. In total, eight transgenic goats were produced.