Expression Of C. Elegans Fat-1 In Rabbit Fetal Fibroblast Cells

Fat-1 gene encodingω-3 polyunsaturated fatty acids(PUFAs) desaturase, can convert polyunsaturated fatty acids fromω6 toω-3 form. co-3PUFAs as an important component of the fatty acids, are concerned by scienists for a long time. It is necessary to maintain the appropriate proportion ofω-3PUFAs that can serve to prevent cardiovascular disease, neurodegenerative diseases and even cancer role. However most animals have to obtain co-3PUFAs from diet, because they cannot synthesize them by themselves. Researchers have used the method of gene transfer to express fat-1 gene in transgenic animals, such as mice, pigs etc that provide efficient and accurate medical, nutritional animal model for the study of co-3PUFAs functions. As the awareness ofω-3PUFAs increasing, the rich ingredients of food demand also increased significantly in people. In the production of special agricultural products, fat-l gene also has very great potential and we need development and utilization.Based on the biased codon usage of rabbit, Caenorhabditis elegans fat-1 cDNA sequence referred from GenBank was optimized and synthesized, fat-1 cDNA was amplified from Caenorhabditis elegans by-RT-PCR in this study. The optimized fat-1 (named as opfat-1) and the amplified fat-1 cDNA were cloned into green fluorescent protein expression vector pEGFP-C1 and transfected to rabbit fetal fibroblast cells using Lipofectamine, respectively. The affecting factors on transfection efficiency, including dose of DNA and liposome as well as exposure time of the cell to the DNA-liposome complexes were compared. The expression of fusion protein and target genes were verified by GFP marker, PCR and RT-PCR assay.The results showed that the optimized transfection condition is 2～3μl Lipofectamine and 0.8-1.0μg DNA for 8h transfection. Transfection efficiency of opfat-1 was significantly evaluated compared with the wild type gene (fat-1 cDNA) by GFP marker. The monoclonal cell lines were obtained by G418 screening. Initial experiments demonstrated fat-1 cDNA and opfat-1 gene were both transcripted steadily in the rabbit fetal fibroblast cells by PCR and RT-PCR.As initial results, this study provided a solid foundation for further research on the expression of functional protein of fat-1 gene and fat-1 transgenic rabbit.