Handmade cloned transgenic sheep rich in omega-3 Fatty acids.
Handmade cloned transgenic sheep rich in omega-3 Fatty acids.
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DOI：
10.1371/journal.pone.0055941
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Ma RZ
中科院分区：
文献类型：
--
作者：
Zhang P;Liu P;Dou H;Chen L;Chen L;Lin L;Tan P;Vajta G;Gao J;Du Y;Ma RZ
体细胞核移植（SCNT）技术已经在世界范围内被用于产生转基因动物，尽管传统的程序主要依赖于仪器显微操作。在这项研究中，我们使用了在牛和猪中建立的改良手工克隆（HMC）来生产omega-3（n-3）脂肪酸水平升高的转基因绵羊。将密码子优化的线虫mfat-1基因插入真核表达载体，并转入中国美利奴绵羊原代成纤维细胞基因组中。进行逆转录酶PCR、气相色谱和染色体分析以选择能够将omega-6（n−6）转化为n−3脂肪酸的核供体细胞。将体外培养7天后发育的囊胚移植到新疆某地方绵羊品种的母绵羊受体子宫内。对于HMC，约8.9%（n = 925）的重构胚胎发育到囊胚期。 将53个囊胚移植到29只自然发情的母羊体内，4只受体妊娠，共获得3只转基因羔羊。对其中一只转基因羔羊的详细分析显示，在绵羊5号染色体上有一个经修饰的线虫mfat-1基因的单一整合。转基因绵羊表达功能性n−3脂肪酸去饱和酶，伴随着肌肉（p<0.01）和其他主要器官/组织（p<0.05）中n−6/n−3比率降低2倍以上。据我们所知，这是HMC生产的转基因绵羊的第一份报告。与传统的SCNT方法相比，HMC显示出相当的效率，但被证明是廉价和易于操作。
Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n−3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n−6) into n−3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n = 925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n−3 fatty acid desaturase, accompanied by more than 2-folds reduction of n−6/n−3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation.
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影响因子：
1.8
作者：
Bhojwani, S;Vajta, G;Poehland, R
通讯作者：
Poehland, R
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3.6
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作者：
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作者：
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通讯作者：
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