Cloned Sheep Derived from Adult Ear Skin Cells

摘要

In the present study, 0.5 % FBS-fed Merino granulosa cells (M-002 cell line) and Poll Dorset ear skin cells (PD-3 and PD-5 cell lines) were used as donor nuclei for nuclear transplantation (NT). A total of 553 oocytes matured in vitro (IVM) were enucleated to produce 447 successfully fused couplets (80%), which resulted in 74 blastocysts following activation using Ionomycin/6-DMAP and culture in SOF media 7 days. Significantly (P < 0.05) more blastocysts developed from the granulosa cell line (26.9%) compared with either the PD-3 (16.5%) or PD-5 (14.9%) skin cell lines. These blastocysts were vitrified using the open-pulled straw (OPS) technique. After 15 month storage in liquid nitrogen, 48 NT blastocysts were thawed and transferred immediately into synchronized 24 recipients resulting in 5 early pregnancies (20%). Two cloned fetuses from the granulosa cell line aborted on Day 85 and Day 133 while three clones from the skin cell lines survived and were delivered to term by cesarean section. Two of these clones died from respiratory problems while the remaining lamb remains alive and healthy. These results indicated that healthy cloned lambs can be bom after long term storage of vitrified cloned embryos.