Production Of Fat-1 Transgenic Bohai Black By Somatic Cell Cloning

The encoded product of fat-1 gene is polyunsaturated fatty acid desaturase. It can convert n-6 polyunsaturated fatty acids (PUFAs) to n-3 PUFAs. In the present study, the MAR sequence mediated monocistronic gene transfer body MAR-CAG-Fat-1-PolyA-MAR was constructed, the Bohai Black Cattle fetal fibroblast cells were transfected, and the transgenic monoclonal cell lines were selected and identified. Transgenic mice were produced by pMAR-CAG-Fat-1-PolyA-MAR vector digested by single enzymes vector) and MAR-CAG-Fat-1-PolyA-MAR vector (digested by two enzymes vector). The results show that:(1) in the transgenic cells, the expressions of fatty acid synthesis associated genes such as FASN, ACC and SREBP-1 were significantly reduced when compared to the controls. The expressions of the fatty acid metabolism associated genes such as PPARA and PPARG decreased. The n-3 PUFAs expression level was increased and n-6/n-3 PUFAs ratio significantly decreased. (2) in the transgenic mice, the n-3 PUFAs expression in muscle and adipose tissues significantly increased in the two enzymes group F0, F1 and F2 transgenic mice, the n-6/n-3 ratio decreased. However, the n-3 PUFAs expression in the muscle and adipose tissues from the single enzyme group transgenic mice decreased, the n-6/n-3 was increased. (3) in the single enzyme group, both of the fatty acid synthesis associated genes FASN, ACC and SREBP-1 and fatty acid metabolism related genes PPARA and PPARG expressed decreased. (4) iTRQ analysis of the single enzyme group showed that 99 different proteins were included, including 62 up-regulated proteins and 37 down-regulated proteins, while 118 different proteins including 81 up-regulated and 37 down-regulated in the two enzyme group have been found. (5) after transfer of transgenic SCNT embryos into the recipient cow, two of the 12 recipients got pregnant and 1 recipient cow developed to term and delivered a healthy calf.In summary, the MAR mediated monocistronic gene transfer body expressed well in both of the transgenic cells and the transgenic mice, and resulted in the production of transgenic cattle.