title: Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance.
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-PGK-neo(r)-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 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.
authors.affiliation: Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Yu Quan Qu, Hohhot, Inner Mongolia, China.
authors.forename: X J
authors.initials: XJ
authors.lastname: Han
authors.affiliation: Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Yu Quan Qu, Hohhot, Inner Mongolia, China.
authors.forename: H
authors.initials: H
authors.lastname: Liang
authors.affiliation: Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Yu Quan Qu, Hohhot, Inner Mongolia, China.
authors.forename: T
authors.initials: T
authors.lastname: Yun
authors.affiliation: Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Yu Quan Qu, Hohhot, Inner Mongolia, China.
authors.forename: Y H
authors.initials: YH
authors.lastname: Zhao
authors.affiliation: State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
authors.forename: M L
authors.initials: ML
authors.lastname: Zhang
authors.affiliation: State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
authors.forename: L H
authors.initials: LH
authors.lastname: Zhao
authors.affiliation: State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China lirongfeng@njmu.edu.cn.
authors.forename: R F
authors.initials: RF
authors.lastname: Li
authors.affiliation: Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Yu Quan Qu, Hohhot, Inner Mongolia, China.
authors.forename: X L
authors.initials: XL
authors.lastname: Li
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D029742:Caenorhabditis elegans Proteins / Q000235:genetics*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics*; D044943:Fatty Acid Desaturases / Q000235:genetics*; D005227:Fatty Acids / Q000032:analysis; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D019426:Integrases; D009355:Neomycin / Q000494:pharmacology*; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D034421:Sus scrofa / Q000196:embryology*
chemical_list: D029742:Caenorhabditis elegans Proteins; D005227:Fatty Acids; C466741:fat-1 protein, C elegans; D012313:RNA; D004247:DNA; D044943:Fatty Acid Desaturases; D010735:Phosphoglycerate Kinase; C045073:Cre recombinase; D019426:Integrases; D009355:Neomycin