Zhang Peng, Zhang Yidi, Dou Hongwei, Yin Jingdong, Chen Yu, Pang Xinzhi, Vajta Gabor, Bolund Lars, Du Yutao, Ma Runlin Z
State Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Cell Reprogram. 2012 Jun;14(3):258-66. doi: 10.1089/cell.2011.0073.
Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals.
通过体细胞核移植（SCNT）生产转基因动物已在全球范围内得到应用，但该应用在一定程度上受到其相对较低效率的限制。在本研究中，我们使用先前建立的手工克隆（HMC）技术来生产表达功能性线虫fat-1基因的转基因猪。将密码子优化的mfat-1插入真核表达载体中，然后将其转入原代猪供体细胞。通过逆转录聚合酶链反应（RT-PCR）、气相色谱法和染色体分析来选择能够将n-6脂肪酸转化为n-3脂肪酸的供体克隆。将n-6/n-3比例降至约1:1的克隆所产生的囊胚通过手术移植到受体猪的子宫内以获得转基因仔猪。通过HMC技术，558个重构胚胎在体外培养7天后，有37%（n = 558）发育到囊胚阶段，平均细胞数为81±36（n = 14）。将408个第6天的囊胚移植到4头自然发情的母猪体内后，3头受体母猪怀孕，共产下14头活仔。线虫mfat-1有效地降低了转基因猪肌肉和主要器官中的n-6/n-3比例。我们的研究结果将有助于建立一种可靠的方法，并为转基因动物的生产提供一种有效的选择。
