畜牧兽医学报 ›› 2009, Vol. 40 ›› Issue (5): 633-638.doi:

• 遗传繁育 • 上一篇    下一篇

体细胞核移植生产转ω-3脂肪酸去饱和酶基因(sFat-1)的猪胚胎

冯冲1,2,周艳荣3,龙川2,刘晓2,陈红星3*,潘登科2*,杨博辉4   

  1. 1. 甘肃农业大学动物科学技术学院,兰州 730070;2. 中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与利用重点实验室,北京 100193; 3. 军事医学科学院生物工程研究所,北京 100071; 4. 中国农业科学院兰州畜牧与兽药研究所,兰州 730050
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-05-25 发布日期:2009-05-25
  • 通讯作者: 潘登科

Production of ω-3 Fatty Acid Desaturase Gene (sFat-1) Transgenic Embryos by Somatic Cell Nuclear Transfer in Pig

FENG Chong1, 2, ZHOU Yan-rong3, LONG Chuan2, LIU Xiao2, CHEN Hong-xing3*, PAN Deng-ke2*, YANG Bo-hui4   

  1. 1. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070,China; 2. The Key Laboratory of Farm Animal Genetic Resources and Utilization of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193,China; 3. Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071,China; 4. Institute of Animal Science and Veterinary Pharmaceutics,Chinese Academy of Agricultural Sciences, Lanzhou 730050,China
  • Received:1900-01-01 Revised:1900-01-01 Online:2009-05-25 Published:2009-05-25

摘要: 本研究通过脂质体介导的方法将来源于线虫C. Briggsae的ω-3脂肪酸去饱和酶基因(sFat-1)转染至大白猪胎儿成纤维细胞。采用600 μg·mL-1 G418药物浓度,经连续10 d筛选及PCR、RT-PCR鉴定,获得11个转基因阳性细胞克隆。以体外成熟42 h的猪卵母细胞与转基因细胞构建重构胚。经体外培养后观察,转基因克隆胚胎与非转基因胚胎的卵裂率 (76.6%±4.1% vs. 81.6%±3.1%) 和囊胚率 (10%±1.97% vs. 9.7%±1.4%) 均无显著差异 (P>0.05)。采用放线菌酮进行化学二次激活时,胚胎的囊胚率显著高于采用电二次激活胚胎 (20.6%±0.89% vs. 10%±1.97%,P<0.05),但二者的卵裂率并无显著差异 (72.4%±4.96% vs. 76.6%±4.1%,P>0.05)。研究表明,通过脂质体介导的方法, 可以获得转sFat1基因大白猪胎儿成纤维细胞系;以该细胞系为核供体构建的转基因克隆胚胎与非转基因克隆胚胎的发育能力无显著差异;二次激活采用放线菌酮进行化学激活能够显著提高胚胎的囊胚发育率。

Abstract: In this study, fetal fibroblast cells of Large White pig were transfected with sFat-1 gene from round-worm C. Briggsae by lipofectamine mediated transfection. After selected by 600 μg·mL-1 G418 for 10 days and analyzed by PCR, RT-PCR, 11 positive transgenic cell clones were collected. Reconstructed embryos were combined by porcine oocytes which were matured for 42 h and transgenic cells. After culture, the cleavage percentage of embryos (76.6%±4.1% vs. 81.6%±3.1%) and blastula (10%±1.97% vs. 9.7%±1.4%) have no significant difference between the transgenic cloning embryos and nontransgenic cloning embryos (P>0.05). In assistant activation, CHX was used and make a higher (20.6%±0.89% vs. 10%±1.97%, P<0.05) percentage of blastula than the electrical activation, but the cleavage percentage was insignificant between them (72.4%±4.96% vs. 76.6%±4.1%, P>0.05). As a result, fetal fibroblast sFat-1 transgenic cell line of Large White pig can be obtained by lipofectamine mediate transfection, and the cell line used as nuclear donors make no significant difference in the development of the transgenic cloning embryos and non-transgenic cloning embryos; Then using CHX in assistant activation can significantly raise the percentage of blastula.