title: Efficient recombinant production in mammalian cells using a novel IR/MAR gene amplification method.
abstract: We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.
authors: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
authors: Yoshio
authors: Y
authors: Araki
authors: Tetsuro
authors: T
authors: Hamafuji
authors: Chiemi
authors: C
authors: Noguchi
authors: Noriaki
authors: N
authors: Shimizu
mesh_terms: D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication / Q000235:genetics*; D004274:DNA, Recombinant / Q000096:biosynthesis* / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics; D045170:Matrix Attachment Regions / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D007074:Immunoglobulin G