利用改进的手工克隆技术生产转GFP基因猪克隆胚胎

doi:10.3724/SP.J.1005.2011.00527

遗传 ›› 2011, Vol. 33 ›› Issue (5): 527-532.doi: 10.3724/SP.J.1005.2011.00527

利用改进的手工克隆技术生产转GFP基因猪克隆胚胎

张鹏^1,2, 杨珍珍², 窦红伟³,李伟杭³, 律波³, Bolund Lars², 杜玉涛^2,3, 谭萍萍¹, 马润林¹

1. 中国科学院遗传与发育生物学研究所, 北京 100101 2. 深圳华大基因研究院, 深圳 518083 3. 深圳华大方舟生物技术有限公司, 深圳 518083

收稿日期:2010-05-27 修回日期:2010-07-09 出版日期:2011-05-20 发布日期:2011-05-25
通讯作者: 杜玉涛 E-mail:duyt@genomics.org.cn
基金资助:
农业部动物转基因重大专项课题(编号: 2009ZX08008-005B)资助

Production of porcine blastocysts expressed EGFP by handmade cloning

ZHANG Peng^1,2, YANG Zhen-Zhen², DOU Hong-Wei³, LI Wei-Hang³, LV Bo³, Bolund Lars², DU Yu-Tao^2,3, TAN Ping-Ping¹, MA Run-Lin¹

1. Institute of genetics and developmental biology, Chinese Acdemy of Science, Beijing 100101, China 2. Beijing Genomics Institute (BGI-ShenZhen), Shenzhen 518083, China 3. BGI ARK Biotechnology Co., Ltd, Shenzhen 518083, China

Received:2010-05-27 Revised:2010-07-09 Online:2011-05-20 Published:2011-05-25
Contact: DU Yu-Tao E-mail:duyt@genomics.org.cn

摘要/Abstract

摘要： 通过体细胞核移植(Somatic cell nuclear transfer, SCNT)培育转基因动物新个体是当前被广泛使用的技术之一, 但其生产成本高和转基因囊胚形成率低在很大程度上制约了该技术的应用。文章报告对该技术的一些改进以提高其成功率并降低成本。首先将增强型绿色荧光基因(EGFP)导入猪胎儿成纤维细胞中, 通过荧光观察EGFP的表达来筛选适合做细胞核移植的体细胞。这样避免了外源EGFP基因虽已整合至猪基因组但不表达的情况, 保证供体细胞100%是表达目标蛋白(绿色荧光蛋白)的细胞; 然后利用新一代体细胞核移植技术——手工克隆技术(Handmade cloning, HMC)将供体细胞与卵母细胞融合生产胚胎。共收集了4个批次378个肉用家猪的卵母细胞, 经体外培养成熟后手工去核得到266个去核卵母细胞, 与EGFP细胞融合后获得127个重构胚胎, 将重构胚胎体外培养到144 h, 得到转基因囊胚65个, 平均囊胚率为52.1±8.3%。与传统SCNT相比, HMC不仅操作简便, 而且能大幅提高核移植细胞的囊胚率。更为重要的是, 改进的手工克隆技术摆脱了昂贵的显微操作仪, 为产业化生产转基因动物提供了新的实用基础。

关键词: 手工克隆, 体细胞核移植, 绿色荧光蛋白, 克隆胚胎

Abstract: Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been widely used worldwide. However, the application of SCNT is impeded by overall high costs and low efficiency. Here, we reported a modification of the existing technology in order to overcome some of the disadvantages associated with SCNT. Firstly, a marker gene, enhanced green fluorescent gene (EGFP), was transfected into pig fetal fibroblast cells, and was subsequently screened by fluorescent expression to ensure donor cells expressing EGFP. Porcine embryos expressing EGFP were then produced by a method called handmade cloning (HMC), a simplified method for micromanipulation. To demonstrate the concept, we col-lected a total of 378 fresh swine oocytes, from which 266 with the nucleus removed, obtained a total of 127 viable recombinant oocytes after fusion with EGFP-expressing cells. In vitro incubation of the 127 recombinant oocytes for approximately 144 hours resulted in successful generation of 65 viable embryos, with an average success rate of 52.1±8.3%. Compared with the traditional SCNT, the method of HMC is not only easy to operate, but also increases the rate of recombinant embryo significantly. Furthermore, the modified method no longer relies on expensive instrument like micromanipulator, facilitating the industrialization of transgenic animal production.

Key words: handmade cloning, SCNT, EGFP, porcine blastocyst

张鹏，杨珍珍，窦红伟，李伟杭，律波，Bolund Lars，杜玉涛，谭萍萍，马润林. 利用改进的手工克隆技术生产转GFP基因猪克隆胚胎[J]. 遗传, 2011, 33(5): 527-532.

ZHANG Feng, YANG Zhen-Zhen, DOU Gong-Wei, LI Wei-Hang, LV Bei, Bolund Lars, DU Yu-Chao, TAN Ping-Ping, MA Run-Lin. Production of porcine blastocysts expressed EGFP by handmade cloning[J]. HEREDITAS, 2011, 33(5): 527-532.

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利用改进的手工克隆技术生产转GFP基因猪克隆胚胎

Production of porcine blastocysts expressed EGFP by handmade cloning

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