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-1, 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 epithelial cells-C127 were transfected with the polyciston transgene vector and then selected using G418 to harvest positive cell clones. The expression of fat-1, 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 fibroblast 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 polyunsaturated fatty acids in milk.