Research Article

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

Published: September 28, 2015
Genet. Mol. Res. 14 (3) : 11594-11604 DOI: 10.4238/2015.September.28.11

Abstract

The neomycin-resistance (neor) 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-neor-hfat-1 expression vector, in which PGK-neor was flanked by two LoxP sites. The pC-PGK-neor-hfat-1 plasmids were transfected into porcine fetal fibroblasts using liposomes, and three transgenic cell lines were obtained by culturing with 400 μg/mL G418 for 7 days. Next, these cell lines were transfected with a Cre recombinase expression plasmid, which contains a puromycin resistance gene, in order to delete neor, which was integrated into the genome. hFat-1-neor 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-neor decreased hFat-1 expression.

The neomycin-resistance (neor) 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-neor-hfat-1 expression vector, in which PGK-neor was flanked by two LoxP sites. The pC-PGK-neor-hfat-1 plasmids were transfected into porcine fetal fibroblasts using liposomes, and three transgenic cell lines were obtained by culturing with 400 μg/mL G418 for 7 days. Next, these cell lines were transfected with a Cre recombinase expression plasmid, which contains a puromycin resistance gene, in order to delete neor, which was integrated into the genome. hFat-1-neor 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-neor decreased hFat-1 expression.