Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance

Abstract

ABSTRACT. The neomycin-resistance (neo r ) gene is widely used as a selectable marker in eukaryotic expression vectors; however, its expression often affects that of target genes. Cre recombinase recognizes LoxP sites, leading to site-specific recombination and deletion of DNA and RNA between two LoxP sites. In the present study, a humanized Fat-1 gene (hFat-1) was generated by DNA Works and used to construct a pC-PGK-neo r -hfat-1 expression vector, in which PGK-neo r was flanked by two loxP sites. The pC-PGKneo r -hfat-1 plasmids were transfected into porcine fetal fibroblasts using liposomes, and three transgenic cell lines were obtained by culturing with 400μg/mL G418 for 7 days. Next, these cell lines 11595 PGK-neo r deletion in hFat-1 transgenic porcine fetal fibroblasts ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (3): 11594-11604 (2015) were transfected with a Cre recombinase expression plasmid, which contains a puromycin resistance gene, in order to delete neo r , which was integrated into the genome. hFat-1-neo r negative cells were obtained following puromycin selection. Real-time quantitative polymerase chain reaction data indicated that neomycin-resistant cells had higher hFat-1 expression than neomycin-sensitive cells. High performance gas chromatography data suggested that the n-6/n-3 ratio was significantly lower in transfected cells than in wild-type cells. The n-6/n-3 ratio in Cre-treated hFat-1-transfected cells was higher than that in untreated cells, suggesting that deletion of PGK-neo r decreased hFat-1 expression.