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

OPIZZO, BIANCA A.; LUCHESSI, GLORIA I.; HEREDIA, ROMINA M.; KUES, WILFRIED; FILI, ALEJANDRO; FORCATO, DIEGO; BOSCH, PABLO

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencia; 2019

Sociedad Argentina de Biología

Vertebrates are unable tosynthesize a subset of polyunsaturated fatty acids (PUFA) known asomega 3 and omega 6. The nematode C.elegans is able tosynthesize them thanks to a family of lipid desaturases (deltadesaturases, i.e., FAT2). Our hypothesis proposes that heterologousexpression of a FAT2 in a bovine mammary gland cell line (MAC-T) willinduce synthesis of PUFA. The aim of the present work was totranspose the C. elegans fat2gene into the genome MAC-T and to study the resulting PUFA profile.Cotransfections of MAC-T with the Sleeping Beauty (SB) transposonsystem were performed. Two transposons, one carrying a cassette forthe expression of a GFP, and a second one for expression of the FAT2enzyme and neomycin resistance were used. For transfection essays,2:0.5:0.5 molar ratios of FAT2, GFP transposons and SB-helper plasmidwere used. After cotransfection, MAC-T cells were subjected toantibiotic selection (G418). After 15 days, fluorescent and resistantcolonies were isolated and expanded. PCR confirmed presence of theFAT2 sequence in five clonal cell lines. Two transgenic cell linesand one unmodified cell clone were grown to confluence in order toanalyze the profile of the cellular phospholipid. Gas chromatographyanalysis of phospholipids confirmed the presence of linoleic acid(C18:2) in both transgenic cell lines, and the absence of linoleicacid in the unmodified cell line. The results indicate thatrecombinant FAT2 is functional, since it catalyzed the synthesis oflinoleic acid, an omega-3 FA. Experiments are ongoing in order toconfirm FAT2-mediated production of longer omega-3 and omega-6 lipids(C20 and C22), which are derived from C18:2. In conclusion, wesuccessfully use the SB transposon system to generate stabletransgenic bovine cell lines that express functional recombinant FAT2enzyme. These results pave the way for the production of geneticallyengineered animals with improved PUFA profiles in tissues or milk forhuman consumption.p { margin-bottom: 0.1in; direction: ltr; color: #000000; line-height: 115%; text-align: left; orphans: 2; widows: 2 }p.western { font-family: "Liberation Serif", serif; font-size: 12pt; so-language: en-US }p.cjk { font-family: "Liberation Serif"; font-size: 12pt; so-language: en-US }p.ctl { font-family: "Liberation Serif"; font-size: 12pt; so-language: ar-SA }