转GFP基因核移植牛胚胎的研究

›› 2009, Vol. 36 ›› Issue (8): 73-78.

转GFP基因核移植牛胚胎的研究

邓守龙¹，吕自力²，王亮^2,4，王安江^1,2，曹文广^3

(1.新疆农业大学动物科学学院，乌鲁木齐 830052； 2.新疆金牛生物有限公司，乌鲁木齐 830026； 3.中国农业科学院北京畜牧兽医研究所，北京 100193； 4.中国科学院新疆理化技术研究所，乌鲁木齐 830011)

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-08-20 发布日期:2009-08-20
通讯作者: 曹文广

Study on Transgenic GFP Gene Bovine Embryos Production by Nuclear Transfer

DENG Shou-long¹, LV Zi-li², WANG Liang^2,4, WANG An-jiang^1,2, CAO Wen-guang³

1.College of Animal Science, Xinjiang Agriculture University, Urumqi 830052, China; 2.XinJiang Jinniu Biological Ltd, Urumqi 830026, China; 3. Chinese Academy of Agricultural Sciences, Beijing Institute of Animal Sciences and Veterinary Medicine, Beijing 100193,China; 4.Chinese Academy of Sciences, Xinjiang Institute of Chemistry and Physics, Urumqi 830011, China)

Received:1900-01-01 Revised:1900-01-01 Online:2009-08-20 Published:2009-08-20
Contact: CAO Wen-guang

摘要/Abstract

摘要： 试验采用脂质体转染法与电穿孔法，以携带绿色荧光蛋白（GFP）-新霉抗性（neo-）双标记基因的pMSCV质粒转染胎牛耳成纤维细胞为供体与体外成熟的牛卵母细胞为受体构建克隆胚。研究了体外成熟培养液中添加EGF（表皮生长因子）对转基因胚的影响，不同转染方法构建供体细胞对重构胚发育的影响和在不同体外培养系统中的发育效果。结果显示，体外成熟培养液中添加EGF 30 ng/mL组的卵母细胞成熟率最高，但对后期转基因重构胚的囊胚发育率的影响，以添加EGF 20 ng/mL组的最高；以胎牛耳成纤维细胞为供体细胞，不同转染方法转染供体细胞构建重构胚，其囊胚发育率差异不显著（P>〖JP2〗0.05）；mSOFaa+颗粒细胞单层细胞共培养体系中的转基因囊胚发育率最好，该体系更适合体细胞核移植法生产转基因牛胚胎。

关键词: 牛; 转基因; 供体细胞转染; 体外培养

Abstract: pMSCV containing GFP and neo- gene was transfected into the cattle fetal auris fibroblast cells by lipofection or electrofusion was the donor. The maturation of bovine oocyte in vitro was receptor. The embryo cloning was constructed by the donor and receptor. Under different conditions,such as adding epidermal growth factor(EGF)to the culture medium improved the nuclear maturation and transgenic embryos in vitro culture, donor cells gene-transfection method, conducted to investigate the effect of three in vitro culture systems on the development of bovine embryos and to observe the development of bovine transgenic embryos in vitro in the optimal culture system will be studied. To examine the effects of EGF on the nuclear and cytoplasm maturation of cattle oocytes in vitro culture, the best concentration of EGF was 30 ng/mL, then the oocytes were activated by 6-dimethylaminopurine combined with ethanol, examining the cleavage rate, blastocyst development rate. The cleavage rate and blastocyst development rate adding 25 ng/mL EGF to medium group were significantly higher than those in control group; and its effect on cell proliferation after grafting. The different gene-transfection method was beneficial to transfer embryo. The rate of the integration was the blastocyst development rate of cattle oocytes significantly（P>0.05）; the highest mSOFaa+granulosa cell monolayer co-culture system. 

Key words: cattle; transgenic; donor cells gene-transfection; in vitro

中图分类号:

S814.8

邓守龙;吕自力;王亮;王安江;曹文广. 转GFP基因核移植牛胚胎的研究[J]. , 2009, 36(8): 73-78.

DENG Shou-long;LV Zi-li;WANG Liang;WANG An-jiang;CAO Wen-guang. Study on Transgenic GFP Gene Bovine Embryos Production by Nuclear Transfer[J]. , 2009, 36(8): 73-78.

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