Lentiviral Gene Transfer Of Sfat-1 Optimizes Fatty Acid Composition In Bovine Fetal Fibroblasts

From epidemiology to cell culture and animal studies to randomized controlled trials, the cardioprotective effects of polyunsaturated fatty acids (PUFAs) are becoming recognized. Recently, Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have been the subject of increasing investigation and have attracted considerable interest as pharmaceutical and nutraceutical compounds. They are subtribe PUFAs and essential components requered for normal cellular function and have been shown to exert many preverntive and therapeutic actions.An important class of enzymes involved in the synthesis of PUFAs is the fatty acid desaturases, which catalyze the introduction of double bonds into the hydrocarbon chain at a position determined by the enzyme specificity.The Caenorhabditis.briggsae sfat-1 gene, which is absent in most animals, encodes anω-3 fatty acid desaturase that convertsω-6 PUFAs toω-3 PUFAs. The objection of current study was the first attempt to transfer sfat-1 gene with lentivirus vectors in the aim to generate transgenic bovine fetal fibroblasts capable of convertingω-6 PUFAs toω-3 PUFAs.To heterologously express the n-3 fatty acid desaturase in early bovine fetal fibroblasts, the sfat-1 gene encoding this protein by optimization of codon usage for mammalian cells was modified and was coupled to a pCMV enhancer/chickenβ-actin promoter (which allows high-level and broad expression of the transgenic in all serieses of bovine fibroblasts). The full length sfat-1 gene was constructed by chemical synthesis in vitro by PCR amplification and restriction ligation.Lentiviral expression plasmid vectors containing GFP domains carrying the sfat-1 gene fragments (pGCLV-sfat1-GFP group) were constructed. Lentivirus with GFP (pGCLV-GFP gourp) domains was performed as a control. After sfat-1 gene fragment transfered, pGCLV-sfat1-GFP group cells and pGCLV-GFP group cells were examined via green fluorescent protein detection. RT-PCR and Western blotting were performed to identify the fusion protein. GC/MS was conducted to analysis the composition of fatty acid by using the extracted total cellular lipids.Lentivirus-mediate sfat-1 had alterated the composition of fatty acid in Chinese Simmental bovine fetal fibroblasts significantly. Heterologous expression of the sfat-1 gene in bovine fetal fibroblasts capable of converting variousω-6 PUFAs to the correspondingω-3 PUFAs, and changed the n-6/n-3 ratio from about 15.32:1 to 0.83:1.This study has demonstrated clearly that the sfat-1 gene can be expressed functionally in bovine fetla fibroblasts, and its expression could confer cells'capability of convertingω-6 PUFAs to correspondingω-3 PUFAs, leading to a balanced n-6/n-3 ratio and a change in fatty acids production. The rearch provides an effective approach to modifying fatty acid composition of bovine fetal fibroblasts and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.