title: High levels of human recombinant cyclooxygenase-1 expression in mammalian cells using a novel gene amplification method.
abstract: We report the expression of a high level of human cyclooxygenase-1 (hCOX-1) in mammalian cells using a novel gene amplification method known as the IR/MAR gene amplification system. IR/MAR-plasmids contain a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) and amplify autonomously without a specific induction process. In this study, the IR/MAR-plasmid pΔBN.AR1 was cotransfected with pCAG-COX1, which expresses hCOX-1, into human HEK293T cells, and G418 and blasticidin S double-resistant cells were obtained in about 1month. Real-time PCR and Western blotting revealed that the expressions of hCOX-1 mRNA and protein in both polyclonal and monoclonal cells were remarkably higher than those in only pCAG-COX1-transfected control cells. Southern blotting demonstrated the amplification of the hCOX-1 gene, and the copy number of clone #43 obtained by the cotransfection of pΔBN.AR1 and pCAG-COX1 was more than 20 copies per cell, though that of clone #14 obtained without using the IR/MAR plasmid pΔBN.AR1 was only two copies. These results indicate that a high level of hCOX-1 expression was achieved as a result of hCOX-1 gene amplification. Furthermore, the crude extract from clone #43 showed a strong COX-1 activity, and the activity was inhibited by the representative COX-1 inhibitor indomethacin, with an IC(50) value of 36nM. These results demonstrate that the IR/MAR gene amplification system is a simple but useful method for generating highly productive mammalian cells.
authors.affiliation: Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co. Ltd., Saitama-shi, Saitama, Japan. hi.yoshimura@po.rd.taisho.co.jp
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Yoshimura
authors.forename: Shingo
authors.initials: S
authors.lastname: Sekine
authors.forename: Hisashi
authors.initials: H
authors.lastname: Adachi
authors.forename: Yoshikatsu
authors.initials: Y
authors.lastname: Uematsu
authors.forename: Akiko
authors.initials: A
authors.lastname: Mitani
authors.forename: Nobuko
authors.initials: N
authors.lastname: Futaki
authors.forename: Noriaki
authors.initials: N
authors.lastname: Shimizu
mesh_terms: D002460:Cell Line; D051545:Cyclooxygenase 1 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D005784:Gene Amplification; D015870:Gene Expression; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
chemical_list: D016861:Cyclooxygenase Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051545:Cyclooxygenase 1; D007213:Indomethacin