Construction Of Complete Sfat-1 Eukaryotic Expression Vector And Establishment Of Transfected Cell Lines

n-3 polyunsaturated fatty acids (PUFAs) that is an important component of the fatty acid, are concerned by scienists for a long time. It is necessary to maintain the appropriate proportion of n-3 PUFAs physiological and it can serve to prevent cardiovascular disease, neurodegenerative diseases and even cancer role. However most animals have to obtain n-3 PUFAs from diet, because they can not synthesize them by themselves. Fat-1 gene, translating into n-3 PUFA desaturase, can convert PUFAs from n-6 to n3 form. Researchers have used the method of gene transfer to the fat-1 gene expression in transgenic animals, such as: mice, pigs for the study of n-3 polyunsaturated fatty acids to provide the functions of an efficient and accurate medical, nutritional animal model. As the awareness of n-3 polyunsaturated fatty acids increasing,the rich ingredients of food demand also increased significantly in people. In the production of special agricultural products,fat-1 gene also has very great potential and we need development and utilization. The nematode gene sfat-1 which optimized the codon is used in this study, we use lipid-transfer method to obtain sfat-1 gene transfered Holstein cow cells, and apply it to cloning research.We synthetic sfat-1 gene using PCR-restriction method. Then reconstruct the gene vector pCDNA3.1(+)-sfat-1-EGFP and negative control vector pCDNA3.1(+)-EGFP based on the Construction of eukaryotic expression vector pCDNA3.1(+).The 8079 TE cell line is transferred using cationic lipid-transfer method, and the positive transgenic cells are obtained after G418 selection. After testing, the PCR, RT-PCR test proved that foreign genes has been integrated within the genome.As initial results, construction of the pCDNA3.1 (+)-EGFP vector is a good negative control in transgenic rearch in my lab.. The establishment of the 8079 TE cell lines can be good donors in variety of transgenic cloned research.Obtaining of sfat-1 gene transfected positive cells lays the foundation for research of n-3 polyunsaturated fatty acids biology at cell level and sfat-1 transgenic cattle.