奶牛体细胞核移植胚胎产业化生产条件优化

生物技术通报 ›› 2013, Vol. 0 ›› Issue (2): 86-92.

奶牛体细胞核移植胚胎产业化生产条件优化

孙伟¹ 巴特尔¹ 郭继彤¹ 李荣凤² 王建国² 李明¹ 胡树香¹ 王春生¹ 李喜和^1,3

（1. 内蒙古赛科星繁育生物技术股份有限公司，呼和浩特 011517; 2. 内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室，呼和浩特 010021; 3. 内蒙古大学蒙古高原动物遗传资源研究中心，呼和浩特 010021）

收稿日期:2012-08-03 修回日期:2013-02-27 出版日期:2013-02-26 发布日期:2013-02-27
作者简介:孙伟，男，硕士，助理研究员，研究方向：动物生殖生物学与生物技术; E-mail ：swzyh769500@163.com
基金资助:
内蒙古自治区“呼和浩特市科技计划项目”（2010-农-6）

Optimization of Production Conditions for Dairy Cows Somatic Cell Nuclear Transfer Embryos

Sun Wei¹ Ba Teer¹ Guo Jitong¹ Li Rongfeng² Wang Jianguo² Li Ming¹ Hu Shuxiang¹ Wang Chunsheng¹ Li Xihe^1,3

（1. Inner Mongolia Saikexing Reproductive Biotechnology Co.，Ltd，Huhhot 011517 ；2. The Key Laboratory of Mammalian Reproductive Biology and Biotechnology of the Ministry of Education，Inner Mongolia University，Hohhot 010021 ；3. Institute for Animal Genetic Resources of Mongolia Plateau Inner Mongolia University，Huhhot 010021）

Received:2012-08-03 Revised:2013-02-27 Published:2013-02-26 Online:2013-02-27

摘要/Abstract

摘要： 通过优化高产奶牛体细胞克隆胚胎体外生产技术条件，制备高质量的奶牛克隆胚胎，旨在提高奶牛体细胞核移植产业化应用效率。就受体卵母细胞去核方法、不同年龄供体牛细胞来源、血清饥饿与否以及不同气相组成培养等条件对奶牛体细胞克隆胚胎生产效率的影响进行了研究和探讨。结果表明，虽然荧光染色辅助去核和盲吸法的去核率、囊胚发育率分别为100%、24.83% 和92.44%、28.26%，两者之间无显著差异（P>0.05），但盲吸法操作简单、效率高；不同年龄来源供体牛的细胞系构建的克隆胚胎的囊胚发育率分别为31.43%、25.68%，两者之间没有显著差异（P>0.05）；经血清饥饿和未饥饿供体细胞重构的克隆胚胎囊胚发育率分别为24%、29.9%，两者之间没有显著差（P>0.05）；富氧和低氧气相培养的克隆胚胎的囊胚发育率分别为28.26%、31.55%，两者之间差异不显著（P>0.05），低氧气相组成更有利于囊胚的发育。根据上述结果，奶牛体细胞核移植胚胎（克隆胚胎）的产业化生产条件为：供体细胞无需进行同期饥饿处理，直接注入到盲吸去核后的受体卵子透明带下构建克隆胚胎，融合后的克隆胚胎在密封的混合三气（5% CO2-5% O2-90% N2）的气相组成下进行体外培养，能保持稳定的囊胚发育率。

关键词: 奶牛, 体细胞, 去核, 核移植, 重构胚胎

Abstract: The purpose of the study was to improve the total industrial application efficiency of dairy cow somatic cell nuclear transfer （SCNT）by optimazing the in vitro producting conditions of dairy cow SCNT embryos. In this study, the effect of different factors, such as enucleation method, sources of donor cell different ages cows, serum starvation, and gas component condition, on the efficiency of SCNT were studied. The results demonstrated that there were no significant differences on the rate of enucleation and the rate of blastocyst（100% and 24.83% in the fluorescent-assistant group and 92.44% and 28.26% in the blind-suction group, respectively）（P>0.05）. However, the method of blind-suction was easier to operate and more efficiency. The age of donor cows showed no significant impact on the rate of blastocyst（31.43% and 25.68%, respectively）. There was no significant difference on the rate of blastocyst between serum-starvation and non-starvation group（24% and 29.9%, respectively）, and there was no significant difference on the rate of blastocyst between high-oxygen and low-oxygen group（28.26% and 31.55%, repsctively）. Conclusionly, the results showed that the optimized condition for industrial production of dairy cow SCNT embryos was as follows ：donor cells without serum-starvation could be injected into ooplasm enucleated by blind-suction to construct cloned embryos. Then, fused cloned embrys should be cultured under sealed condition with 5% CO2, 5% O2, and 90% N2 to get a reasonable rate of blastocyst.

Key words: Dairy cows, Somatic cell, Ennuclear, Nuclear transfer, Reconstructed embryos

孙伟, 巴特尔, 郭继彤, 李荣凤, 王建国, 李明, 胡树香, 王春生, 李喜和. 奶牛体细胞核移植胚胎产业化生产条件优化[J]. 生物技术通报, 2013, 0(2): 86-92.

Sun Wei, Ba Teer, Guo Jitong, Li Rongfeng, Wang Jianguo, Li Ming, Hu Shuxiang, Wang Chunsheng, Li Xihe. Optimization of Production Conditions for Dairy Cows Somatic Cell Nuclear Transfer Embryos[J]. Biotechnology Bulletin, 2013, 0(2): 86-92.

参考文献

［1] Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells［J]. Nature, 1997, 385 (6619)：810-813.
[2] Baguisi A, Behboodi E, Melican AD, et al. Production of goats by somatic cell nuclear transfer［J].Nature Biotech, 1999, 17(5)： 456-461.
[3] Cibelli JB, Stice SL, Golueke PJ, et al. Cloned transgenic calves produced from nonquiescent fetalfibroblasts［J]. Science, 1998, 280(5367)：1256-1258.
[4] Betthauser J, Forsberg E, Augenstein M, et al. Production of cloned pigs from in vitro systems［J]. Nature Biotech, 2000, 18(10)： 1055-1059.
[5] Wakayama T, Yanagimachi R. Cloning of male mice from adult tailtip cells［J]. Nat Genet, 1999, 22(2)：127-128.
[6] Galli C, Lagutina I, Crotti G, et al. Pregnancy:a cloned horse born to its dam twin［J]. Nature, 2003, 424(6949)：635.
[7] Wani NA, Wernery U, Hassan FA, et al. Production of the first cloned camel by somatic cell nuclear transfer［J]. Biology of Reproduction, 2010, 82(2)：373-379.
[8] Berg DK, Li CY, Asher G, et al. Red Deer cloned from antler stem cells and their differentiated progeny［J].Biology of Reproduction, 2007, 77(3)：384-394.
[9] RideoutIII WM, Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome［J]. Science, 2001, 293 (5532)：1093-1098.
[10] 郝振华, 伊璐, 刘红林, 等. 哺乳动物细胞核编程方法学研究进展［J]. 畜牧与兽医, 2007(12)：71-75.
[11] 张小建, 李键, 王蕊, 等. 供体细胞对哺乳动物体细胞核移植的影响［J]. 动物医学进展, 2007, 28(3)：82-85.
[12] Vignon X, LeBourshis D, Chesne P, et al. Development of bovine nuclear transfer embryos reconstituted with quiescent and proliferative skin fibroblasts［J]. Heriogenology, 1999, 51:216. 生物技术通报 Biotechnology Bulletin 2013年第2期92
[13] Dominko T, Mitalipova M, Haley B, et al. Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species［J]. Biol Reprod, 1999, 60:1496-502.
[14] Blelloch R, Wang Z, Meissner A, et al. Reprogramming efficiency following somatic cell nuclear transfer is influenced by the differentiation and methylation state of the donor nucleus［J]. Stem Cell, 2006, 24:2007-213.
[15] 黄国宁, 孙海翔. 体外受精-胚胎移植实验室技术［M]. 北京：人民卫生出版社, 2012:208-209.
[16] Varisanga MD, Dong YJ, Mtango NK, et al. Comparison of the effects of using standard and simple portable CO2 incubators on the in vitro developmental competence of bovine embryos reconstituted by somatic cell nuclear transfer［J]. Theriogenology, 2002, 58: 77-86.
[17] Machaty Z, Day BN, Prather RS. Development of early porcine embryos in vitro and in vivo［J]. BiloReprod, 1998, 59(2)： 451-455. (责任编辑李楠)

[1]	曾虹, 曾睿琳, 付伟, 吉文汇, 兰道亮. 牛诱导多能干细胞的建立及应用研究进展[J]. 生物技术通报, 2023, 39(5): 130-141.
[2]	李宇航, 王兴平, 杨箭, 罗仍卓么, 任倩倩, 魏大为, 马云. miR-665在奶牛乳腺上皮细胞炎症中的表达及功能分析[J]. 生物技术通报, 2022, 38(5): 159-168.
[3]	王晋鹏, 罗仍卓么, 王兴平, 杨箭, 贾立, 马云, 魏大为. 奶牛乳腺炎治疗及抗炎分子机制的研究进展[J]. 生物技术通报, 2021, 37(12): 212-219.
[4]	张萌, 罗芳, 王敏, 武彦泽, 王俊奎, 和东迁, 陈丽尧, 陶金忠. 奶牛分娩后早期血浆代谢物变化研究[J]. 生物技术通报, 2020, 36(6): 191-199.
[5]	杨佳怡, 牛春艳, 刘瑛颖, 傅博强, 王晶. 生鲜牛乳体细胞检测与计量校准必要性研究[J]. 生物技术通报, 2020, 36(5): 16-21.
[6]	吴家劲, 朱森林, 周密, 孙会增. 奶牛瘤胃微生物研究进展和趋势[J]. 生物技术通报, 2020, 36(2): 27-38.
[7]	胡启超, 罗仍卓么, 魏大为, 杨箭, 贾立, 王兴平, 马云. 固有免疫相关编码基因在奶牛乳腺炎调节中的研究进展[J]. 生物技术通报, 2020, 36(12): 239-246.
[8]	张萌, 刘国林, 李向龙, 陈永宏, 白玲荣, 罗芳, 李亚超, 陶金忠. 围产前期添加山楂和黄芪混合物对奶牛血浆代谢组的影响[J]. 生物技术通报, 2019, 35(8): 127-137.
[9]	吴丽芳, 魏晓梅, 陆伟东. 白刺花胚性愈伤组织诱导及体细胞胚发生和萌发[J]. 生物技术通报, 2019, 35(4): 13-19.
[10]	明鹏飞, 黄莹莹, 董妍丽, 聂星灿, 冯士彬, 王希春, 程建波, 李锦春, 吴金节, 李玉. LKB1-AMPKα-SIRT1信号通路在奶牛脂肪组织脂代谢中的调控作用[J]. 生物技术通报, 2019, 35(2): 176-181.
[11]	吴霄, 庄站伟, 马晓莉, 黄思秀, 李紫聪, 徐铮. 核移植介导的哺乳动物体细胞核重编程研究进展[J]. 生物技术通报, 2019, 35(11): 187-194.
[12]	赵方东, 李麟坤, 何旭升, 曾会明. 关键基因和相关激素在植物胚胎发生前后期的作用[J]. 生物技术通报, 2017, 33(12): 30-36.
[13]	张宏燕, 信吉阁. 猪体细胞核移植技术研究进展[J]. 生物技术通报, 2016, 32(8): 41-46.
[14]	奥旭东,萨如拉,王杰,王会敏,于海泉. DNA甲基转移酶抑制剂5-Aza-CdR对AID基因修饰的牛胎儿成纤维细胞的作用[J]. 生物技术通报, 2016, 32(8): 103-112.
[15]	许锴,陈霞,高绍荣. 我国诱导多能干细胞研究进展[J]. 生物技术通报, 2015, 31(4): 72-81.