Oh Byoung-Chol, Kim Jung-Tae, Shin Nam-Shik, Kwon Soo-Whan, Kang Sung-Keun, Lee Byeong-Chun, Hwang Woo-Suk
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, and The Xenotransplantation Research Center, Seoul National University Hospital, Korea.
J Vet Med Sci. 2006 Nov;68(11):1167-71. doi: 10.1292/jvms.68.1167.
Interspecies cloning may be a useful method to help conserve endangered species and to study nuclear-cytoplasm interaction. The present study investigated in vitro development of goral (Naemorhedus goral) intergeneric nuclear transfer embryos produced by fusing goral fibroblasts with enucleated metaphase II (MII) bovine oocytes. After two to five passages, serum-starved or non-starved goral skin fibroblast cells were transferred into enucleated MII bovine oocytes. Couplets were electrically fused and chemically activated, and then cultured in either modified synthetic oviduct fluid (mSOF) or tissue culture medium-199 (TCM-199) supplemented with 10% FBS. Serum starvation of donor cells did not affect the fusion rate and or development to of cells to the two-cell stage, to more than 9-cells, or to morulae, regardless of culture medium. Three blastocysts from 202 fused embryos were obtained when embryos reconstructed with non- serum- starved donor cells were cultured in mSOF. However, no blastocysts were obtained when the embryos reconstructed with serum-starved donor cells were cultured in mSOF. The total cell number of goral intergeneric embryos averaged 130.3 (range 105-180). In conclusion, this study demonstrated that bovine oocytes can support blastocyst development after intergeneric SCNT with goral fibroblasts.
种间克隆可能是一种有助于保护濒危物种和研究核质相互作用的有用方法。本研究调查了通过将斑羚成纤维细胞与去核的中期 II（MII）牛卵母细胞融合产生的斑羚属间核移植胚胎的体外发育情况。经过两到五次传代后，将血清饥饿或未饥饿的斑羚皮肤成纤维细胞转移到去核的 MII 牛卵母细胞中。将细胞对进行电融合和化学激活，然后在添加有 10%胎牛血清的改良合成输卵管液（mSOF）或组织培养基 199（TCM-199）中培养。无论培养基如何，供体细胞的血清饥饿都不会影响融合率以及细胞发育到二细胞期、超过九细胞期或桑葚胚期。当用未血清饥饿的供体细胞重建的胚胎在 mSOF 中培养时，从 202 个融合胚胎中获得了三个囊胚。然而，当用血清饥饿的供体细胞重建的胚胎在 mSOF 中培养时，未获得囊胚。斑羚属间胚胎的总细胞数平均为 130.3（范围为 105 - 180）。总之，本研究表明，牛卵母细胞在与斑羚成纤维细胞进行属间体细胞核移植后能够支持囊胚发育。
