Construction of Fat-1 Gene Vector and its Expression in vitro of Chicken

The aim of the experiment was to construct an eukaryotic expression vector which could effectively express ω-3 fatty acid desaturase Fat-1 gene in cells of chicken and laid the foundations for production of transgenic chicken in the future.Based on the codon usage frequency of poultry, nucleotide sequence of Fat-1 gene of Caenorhabditis elegans was optimized (designed as cFat-1), synthesized and connected to pLL3.7 vector.The recombinant plasmid was confirmed by PCR, enzyme digestion and sequence analysis.The chicken embryo fibroblast cells were isolated using standard pancreatic enzyme digestion method and then transfected with pLL3.7-cFat-1 vector by TurboFect^TM transfection reagents in vitro.The gene expression was verified by RT-PCR analysis and green fluorescent detection.The results showed that cFat-1 gene could transcript and express efficiently in chicken embryo fibroblast cells indicating that the Fat-1 gene vector was constructed successfully.It was the first time that transgenic chicken fibroblast cell line which expressed cFat-1 gene was established.This study laid the foundation for the generation of transgenic chicken that were rich in ω-3 fatty acid.