Kwon Dae Kee, Kang Jung Taek, Park Sol Ji, Gomez Ma Ninia Limas, Kim Su Jin, Atikuzzaman Mohammad, Koo Ok Jae, Jang Goo, Lee Byeong Chun
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea.
Zygote. 2011 Aug;19(3):199-204. doi: 10.1017/S0967199411000232. Epub 2011 May 4.
In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus-oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5-5.5% CO2 under various conditions (37-39 °C and 5-20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.
在非人类灵长类动物中，通过体细胞核移植（SCNT）来收集足够数量的卵母细胞以产生相同的胚胎是很困难的。由于这个因素，使用异种卵母细胞的种间体细胞核移植（iSCNT）是一种有吸引力的替代方法。本研究的目的是使用去核的牛（Bos taurus）减数分裂II期卵母细胞和成年恒河猴（Macaca mulatta）成纤维细胞来生产iSCNT来源的囊胚。通过活检收集一只6岁雄性恒河猴的耳部皮肤组织，并分离出成纤维细胞。从牛卵巢中收集未成熟的卵丘-卵母细胞复合体，并在199培养基中进行体外成熟培养。将去核的卵母细胞与恒河猴成纤维细胞进行重构，然后将iSCNT胚胎在改良的合成输卵管液中，于5-5.5%二氧化碳的气氛下，在各种条件（37-39°C和5-20%氧气）下进行培养，以研究体外培养条件的影响。大多数胚胎停滞在8细胞或16细胞阶段，在总共1153个培养的激活胚胎中，通过iSCNT仅获得了3个囊胚（产生率为0.26%）。三个囊胚中的两个用于通过双苯甲酰亚胺染色来计数核数量，分别为51和24。另一个iSCNT来源的囊胚用于通过PCR分析线粒体DNA（mtDNA），检测到了恒河猴和牛的mtDNA。尽管发育率极低，但本研究证实，使用包括灵长类动物在内的两个系统发育距离较远的物种进行iSCNT可以产生囊胚。随着发育率的提高，有可能生产出恒河猴iSCNT来源的胚胎干细胞系，用于研究灵长类细胞核与牛线粒体的相互作用机制。
