Oh H J, Kim M K, Jang G, Kim H J, Hong S G, Park J E, Park K, Park C, Sohn S H, Kim D Y, Shin N S, Lee B C
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
Theriogenology. 2008 Sep 1;70(4):638-47. doi: 10.1016/j.theriogenology.2008.04.032. Epub 2008 Jun 4.
The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.
本研究的目的是调查将死后狼体细胞的细胞核移植到去核犬卵母细胞中是否是生产克隆狼的可行方法。将体内成熟的卵母细胞（来自家犬）去核，并与来自一只雄性灰狼死后6小时获取的组织培养物中的体细胞融合。重构胚胎被激活并移植到自然同步的家犬母犬的输卵管中。总体而言，372个重构胚胎被移植到17只受体犬中；4只受体犬（23.5%）在胚胎移植后23 - 25天经超声检查确认怀孕。一只受体犬自然分娩了两只死胎幼犬，三只受体犬通过剖腹产分娩了四只克隆狼幼犬，体重分别为450克、190克、300克和490克。体重190克的幼犬在出生后12小时内死亡。这六只克隆狼幼犬与供体狼基因相同，它们的线粒体DNA来自卵母细胞供体。三只存活的狼幼犬具有正常的狼核型（78，XY），通过定量荧光原位杂交评估的端粒DNA量与核供体相似或低于核供体。总之，本研究通过将死后从狼身上分离的体细胞跨物种转移到去核犬卵母细胞中，成功克隆了一只濒危雄性灰狼。因此，体细胞克隆技术在包括突然死亡在内的极端情况下，对于保护犬科物种具有潜力。
