Xu Lianguang, Mesalam Ayman, Lee Kyeong-Lim, Song Seok-Hwan, Khan Imran, Chowdhury M M R, Lv Wenfa, Kong Il-Keun
1 Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.
2 Department of Theriogenology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
Cell Reprogram. 2019 Feb;21(1):51-60. doi: 10.1089/cell.2018.0050.
Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.
体细胞核移植（SCNT）是一项有用的技术；然而，其效率较低。在本研究中，我们通过末端脱氧核苷酸转移酶dUTP缺口末端标记、定量逆转录PCR和免疫细胞化学，研究了将细胞质注入去核卵母细胞对克隆的植入前牛胚胎的发育能力和质量的影响。我们使用了细胞质注射克隆技术（CICT），这是一种新技术，通过注入约30%供体卵母细胞的细胞质可以恢复去核卵母细胞的细胞质体积。CICT组胚胎的卵裂率和囊胚形成率显著高于SCNT组（分别为28.9±0.8%和20.2±1.3%，p<0.05）。此外，CICT组第8天囊胚的总细胞数显著高于SCNT组（176.2±6.5对119.3±7.7，p<0.05）。而且，使用MitoTracker Green检测发现CICT增加了线粒体活性。CICT组中DNA甲基转移酶1和DNA甲基转移酶3a的mRNA水平显著低于SCNT组（p<0.05）。CICT组中DNA甲基转移酶3b的mRNA水平低于SCNT组；然而，这种差异不显著（p>0.05）。综上所述，这些数据表明CICT提高了克隆牛胚胎的体外发育能力和质量。
