title: Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.
abstract: Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.
authors.affiliation: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
authors.forename: Chiemi
authors.initials: C
authors.lastname: Noguchi
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Araki
authors.forename: Daisuke
authors.initials: D
authors.lastname: Miki
authors.forename: Noriaki
authors.initials: N
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005784:Gene Amplification; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D011961:Receptors, Fc; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins