乳腺特异性共表达4种脂肪酸合成酶载体及其稳转山羊成纤维细胞系的构建

Construction of mammary gland specific transgene vector harboring four fat acid synthetase genes and establishment of transgenic fibroblast cell lines in goat

为改善山羊乳品质,创制乳汁中富含n-3不饱和脂肪酸的奶山羊育种新材料,首先构建了一种山羊乳腺特异性表达载体,以实现n-3不饱和脂肪酸合通路中4种脂肪酸合成酶基因(fat-1,fads2,elovl5和scd-1)的共表达。为初步验证该载体的有效性,通过脂质体法转染小鼠乳腺上皮细胞系C127,经G418筛选收集阳性C127转基因细胞,并对其进行反转录PCR(RT-PCR)检测。结果表明,4种基因均可在C127细胞转基因中表达。将该多基因共表达载体转染奶山羊成纤维细胞,通过G418筛选获得了稳定转染的细胞系。PCR结果显示,4个目的基因均成功整合到奶山羊成纤维细胞基因组中。总之,本研究成功构建了乳腺特异性共表达4种脂肪酸合成酶转基因载体及其山羊转基因细胞系,为创制乳腺特异性表达多不饱和脂肪酸的转基因奶山羊奠定了基础。

To enrich the content of n-3 polyunsaturated fatty acids and improve the milk quality in goat using transgenic technology, we constructed a mammary gland specific expression vector harboring four key genes （fat-l, fads2, elovl5 and scd-1） encoding key proteins/enzymes that are actively involved in fatty acid synthesis. To validate the structural integrity and functions of the constructed vector, mouse mammary epithe- lial cells-C127 were transfected with the polyciston transgene vector and then selected using G418 to harvest positive cell clones. The expression of fat-l, fads2, elovl5 and scd-1 was analyzed using reverse transcriptional polymerase chain reaction （RT-PCR）. The results revealed that the four genes were expressed as expected, which indicated that the transgene construct is intact and functional in C127 cells. Finally, the goat fibroblast cells were transfected using Fugene HD. The transgenic fibroblast cell lines in goat were obtained after G418 selection. The PCR results showed that the four genes were also successfully integrated into the dairy goat fi- broblast cells. In all, a mammary gland specific vector expressing four key genes for fatty acid synthetase was constructed and the transgenic cell lines containing this polyciston transgene construct in goat were obtained. This research would lay a solid foundation for development of transgenic goats producing enriched polyun- saturated fatty acids in milk.