Expression of recombinant fatty acid desaturase in a bovine mammary gland cell line induces changes in lipid profiles.

OPIZZO, BIANCA A.; LUCHESSI, GLORIA I.; FILI, ALEJANDRO; FORCATO, D.O.; BOSCH, P.; HEREDIA, ROMINA M.; KUES, WILFRIED

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencia; 2019

Sociedad Argentina de Biología

Vertebrates are unable to synthesize asubset of polyunsaturated fatty acids (PUFA) known as omega 3and omega 6. The nematode C. elegans is able to synthesizethem thanks to a family of lipid desaturases (delta desaturases,i.e., FAT2). Our hypothesis proposes that heterologousexpression of a FAT2 in a bovine mammary gland cell line (MACT) will induce synthesis of PUFA. The aim of the present workwas to transpose the C. elegans fat2 gene into the genome MACT and to study the resulting PUFA profile. Cotransfections ofMAC-T with the Sleeping Beauty (SB) transposon system wereperformed. Two transposons, one carrying a cassette for theexpression of a GFP, and a second one for expression of the FAT2enzyme and neomycin resistance were used. For transfectionessays, 2:0.5:0.5 molar ratios of FAT2, GFP transposons and SBhelper plasmid were used. After cotransfection, MAC-T cells weresubjected to antibiotic selection (G418). After 15 days,fluorescent and resistant colonies were isolated and expanded.PCR confirmed presence of the FAT2 sequence in five clonal celllines. Two transgenic cell lines and one unmodified cell clonewere grown to confluence in order to analyze the profile of thecellular phospholipid. Gas chromatography analysis ofphospholipids confirmed the presence of linoleic acid (C18:2) inboth transgenic cell lines, and the absence of linoleic acid in theunmodified cell line. The results indicate that recombinant FAT2is functional, since it catalyzed the synthesis of linoleic acid, anomega-3 FA. Experiments are ongoing in order to confirm FAT2-mediated production of longer omega-3 and omega-6 lipids (C20and C22), which are derived from C18:2. In conclusion, wesuccessfully use the SB transposon system to generate stabletransgenic bovine cell lines that express functional recombinantFAT2 enzyme. These results pave the way for the production ofgenetically engineered animals with improved PUFA profiles intissues or milk for human consumption.