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整合人lactoferrin基因的山羊体细胞支持核移植克隆胚的体外发育

Nuclear Transfer of Goat Somatic Cells Transgenic for Human Lactoferrin

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【作者】 李兰沈伟潘庆玉闵令江孙玉江房勇为邓继先潘庆杰

【Author】 LI Lan1,2, SHEN Wei1,2, PAN Qing-Yu3, MIN Ling-Jiang1, SUN Yu-Jiang4, FANG Yong-Wei1, DENG Ji-Xian2, PAN Qing-Jie1 (1. Institute of Animal Reproduction, Development and Genetic Engineering, Department of Animal Sciences, Laiyang Agriculture College, Qingdao 266109, China; 2. Institute of Biotechnology, Academy of Military Medical Science, Beijing 100071, China; 3.Yantai Fushan Advanced Vocation School, Yantai 265500, China; 4. Institute of Dongying Agricultural Sciences, Dongying 257000, China)

【机构】 莱阳农学院动物科技学院动物生殖发育与基因工程研究所烟台福山高级职业学校东营农业科学研究所军事医学科学院生物工程研究所莱阳农学院动物科技学院动物生殖发育与基因工程研究所 青岛266109 军事医学科学院生物工程研究所北京100071青岛266109 军事医学科学院生物工程研究所烟台265500青岛266109东营257000

【摘要】 克隆了人lactoferrin基因和山羊β-casein基因5′端调控区,构建了人lactoferrin的乳腺表达载体,并将该载体利用脂质体介导转染了奶山羊胎儿成纤维细胞,获得了稳定整合人lactoferrin基因的转基因体细胞克隆17个,其中PCR和SouthernBlot检测阳性的细胞克隆14个,阳性率82.4%。以转基因体细胞为供体细胞进行了核移植,获得了能够体外发育的山羊转基因克隆胚胎,体内成熟卵母细胞来源的核移植囊胚率为64.8%,体外成熟卵母细胞来源的核移植囊胚率为51.7%,证明了山羊转基因体细胞能够支持克隆胚的进一步发育。

【Abstract】 Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat β-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

【基金】 国家高技术研究发展计划项目(国家863计划)(编号:2002AA206621);山东省科技攻关项目(编号:J05K06);莱阳农学院博士启动基金(编号:630615)资助~~
  • 【分类号】Q813
  • 【被引频次】4
  • 【下载频次】155
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