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.

参考文献

[1] Lai LX, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS. Production of α-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science, 2002, 295(5557): 1089-1092.
[2] Dai YF, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL. Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol, 2002, 20(3): 251-255.
[3] Lai LX, Kang JX, Li RF, Wang JD, Witt WT, Yong HY, Hao YH, Wax DM, Murphy CN, Rieke A, Samuel M, Lin-ville ML, Korte SW, Evans RW, Starzl TE, Prather RS, Dai YF. Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol, 2006, 24(4): 435-436.
[4] Park KW, Choi KM, Hong SP, Han GS, Yoo JY, Jin DI, Seol JG, Park CS. Production of transgenic recloned pig-lets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer. Theriogenology, 2008, 70(9): 1431-1438.
[5] Vajta G. Handmade cloning: the future way of nuclear transfer? Trends Biotechnol, 2007, 25(6): 250-253.
[6] Vajta G, Lewis IM, Hyttel P, Thouas GA, Trounson AO. Somatic cell cloning without micromanipulators. Cloning, 2001, 3(2): 89-95.
[7] Vajta G, Bartels P, Joubert J, de la Rey M, Treadwell R, Callesen H. Production of a healthy calf by somatic cell nuclear transfer without micromanipulation and carbon dioxide incubators using handmade cloning (HMC) and submarine incubation system (SIS). Theriogenology, 2004, 62(8): 1465-1472.
[8] Vajta G, Kragh PM, Mtango NR, Callesen H. Hand-made cloning approach: potentials and limitations. Reprod Fertil Dev, 2005, 17(1-2): 97-112.
[9] Tecirlioglu RT, French AJ, Lewis IM, Vajta G, Korfiatis NA, Hall VJ, Ruddock NT, Cooney MA, Trounson AO. Birth of a cloned calf derived from a vitrified hand made cloned embryo. Reprod Fertil Dev, 2003, 15(7-8): 361-366.
[10] Kragh PM, Vajta G, Corydon TJ, Purup S, Bolund L, Callesen H. Production of transgenic porcine blastocysts by hand-made cloning. Reprod Fertil Dev, 2004, 16(3): 315-318.
[11] Kragh PM, Du Y, Corydon TJ, Purup S, Bolund L, Vajta G. Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation. Theriogenology, 2005, 64(7): 1536-1545.
[12] Du Y, Kragh PM, Zhang X, Purup S, Yang H, Bolund L, Vajta G. High overall in vitro efficiency of por-cine handmade cloning (HMC) combining partial zona digestion and oocyte trisection with sequential culture. Cloning Stem Cells, 2005, 7(3): 199-205.
[13] Du Y, Kragh PM, Zhang Y, Li J, Schmidt M, Bøgh IB, Zhang X, Purup S, Jørgensen AL, Pedersen AM, Villemoes K, Yang H, Bolund L, Vajta G. Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology, 2007, 68(8): 1104-1110.
[14] Vajta G, Holm P, Greve T, Callesen H. The submarine incubation system, a novel tool for in vitro embryo culture. Theriogenology, 1997, 48(8): 1379-1385.
[15] Li J, Du Y, Zhang YH, Kragh PM, Purup S, Bolund L, Yang H, Xue QZ, Vajta G. Chemically assisted handmade enucleation of porcine oocytes. Cloning Stem Cells, 2006, 8(4): 241-250.
[16] Yoshioka K, Suzuki C, Tanaka A, Anas IM, Iwamura S. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod, 2002, 66(1): 112-119.
[17] Vajta G, Peura TT, Holm P, Páldi A, Greve T, Trounson AO, Callesen H. New method for culture of zonaincluded or zona-free embryos: the Well of the Well (WOW) sys-tem. Mol Reprod Dev, 2000, 55(3): 256-264.
[18] Feltrin C, Forell F, dos Santos L, Rodrigues

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

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

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	李亚楠, 张贤君, 张宁, 梁雅琳, 张宇星, 招华兴, 李紫聪, 黄思秀. 过表达组蛋白H3K9me3去甲基化酶对猪克隆胚胎发育的影响[J]. 遗传, 2023, 45(1): 67-77.
[2]	宋绍征, 何正义, 成勇, 于宝利, 张婷, 李丹. TALENs介导MSTN基因突变山羊的制备及性能分析[J]. 遗传, 2022, 44(6): 531-542.
[3]	周俊, 赵成成, 吴霄, 石俊松, 周荣, 吴珍芳, 李紫聪. 猪耳成纤维细胞转录组异质性及对核移植胚胎发育的潜在影响[J]. 遗传, 2020, 42(9): 898-915.
[4]	敖政, 陈祥, 吴珍芳, 李紫聪. 体细胞克隆猪发育异常研究进展[J]. 遗传, 2020, 42(10): 993-1003.
[5]	杨旭琼, 吴珍芳, 李紫聪. 哺乳动物体细胞核移植表观遗传重编程研究进展[J]. 遗传, 2019, 41(12): 1099-1109.
[6]	严婷婷, 张蕾, 李余动, 梁新乐. 基于微信的“微生物遗传育种实验”混合式教学模式探究[J]. 遗传, 2018, 40(7): 601-606.
[7]	康岚, 陈嘉瑜, 高绍荣. 中国细胞重编程和多能干细胞研究进展[J]. 遗传, 2018, 40(10): 825-840.
[8]	敖政, 刘德武, 蔡更元, 吴珍芳, 李紫聪. 克隆哺乳动物的胎盘发育缺陷[J]. 遗传, 2016, 38(5): 402-410.
[9]	李智方, 冯冲, 纪慧丽, 石宁宁, 宋小凤, 赵勤丽, 龙川, 潘登科, 杨小淦. 绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析[J]. 遗传, 2015, 37(12): 1211-1217.
[10]	李飞达, 李勇, 刘欢, 张欢欢, 刘楚新, 张兴举, 窦红伟, 杨文献, 杜玉涛. 利用TALENs和手工克隆技术高效获得GHR基因敲除巴马猪[J]. 遗传, 2014, 36(9): 903-911.
[11]	纪慧丽, 卢晟盛, 潘登科. 体细胞核移植后表观遗传重编程的异常及其修复[J]. 遗传, 2014, 36(12): 1211-1218.
[12]	范宗兴，朱化彬，杜卫华. 细胞抽提物诱导的体细胞重编程[J]. 遗传, 2013, 35(3): 262-268.
[13]	孔庆然，朱江，黄波，郇延军，王峰，石永乾，刘仲凤，武美玲，刘忠华. TSA促进转基因猪体细胞核移植胚胎发育和外源基因表达[J]. 遗传, 2011, 33(7): 749-756.
[14]	苏建民，许文兵，李艳艳，王丽君，王勇胜，张涌. 转基因克隆牛胎盘中印迹基因PEG10的DNA甲基化水平[J]. 遗传, 2011, 33(5): 533-538.
[15]	李姗姗，雍静茹，齐云玲，章颖，赵亮，夏士林，李东，王慧利，包其郁，李佩珍. 螺旋藻耐盐相关基因启动子区功能分析[J]. 遗传, 2011, 33(10): 1134-1140.