The method is 3) efficient; almost all the transfected cells bear the amplified target gene, thus the frequency of high-producer cell clones is quite high. 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 OAC2 clone. Introduction Production of recombinant proteins in OAC2 cultured mammalian cells is becoming more critical as the need for large amounts of pharmaceuticals protein, e.g. humanized antibody, is increasing rapidly. Large-scale culture of mammalian cells is more expensive and technically challenging than that of yeast or bacterial cells. However, patterns of protein folding and protein modification, such as glycosylation, are specific to mammalian cells, and bacterial and yeast LAMB3 proteins may invoke immune responses in humans. Furthermore, the presence of trace amounts of yeast or bacterial components in preparations of proteins for therapeutic use is unacceptable. Therefore, proteins for therapeutic use must be produced in mammalian cells. For industrial protein production, the most popular mammalian cell has been the Chinese hamster ovary (CHO) cell line and its derivatives. Industrial production of recombinant protein in these cells is a multi-part process and entails the development of high-producer cells, culture of the cells at high density in chemically defined medium, and purification of the target protein (reviewed in [1]). Here, we OAC2 describe an improvement in the first step of this process with the introduction of a novel gene amplification method that efficiently increases target gene copy number in the cultured cells. Amplification of oncogenes or drug-resistance genes has frequently been associated with the malignant transformation of human cells, where gene amplification induces overproduction of the cognate protein product. Therefore, the induction of target gene amplification has often been used to generate cells that produce high levels of a target for OAC2 the pharmaceutical industry. A frequently used method for target gene amplification is the linkage of the dihyfrofolate reductase (DHFR) gene to the target gene, followed by amplification induced by increasing concentrations of the DHFR inhibitor methotrexate (MTx) in a DHFR-deficient CHO subline, such as DG44. However, this method is time- and labor-intensive [2], usually requiring more than six months for a skilled technician to complete. Furthermore, the high-producer cells produced by this method are frequently unstable [3], and the structural integrity and productivity of the transgene often declines rapidly. Such instability was also reported for another gene amplification-mediated method (GS/MSX method; OAC2 [4], [5]). Therefore, an alternative method that enables rapid and efficient acquisition of stable high-producer cell is strongly required [1]. As an alternative to this approach, we previously developed a new method that amplifies any gene in mammalian cells [6], [7]. The method utilizes a plasmid that has a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR); thus, we refer to the technique as IR/MAR gene amplification. When this plasmid was introduced into human colorectal carcinoma COLO 320 cells, a pool of stable transfectants was obtained after selecting for plasmid-coded drug-resistance to a drug such as blasticidin. Fluorescence in situ hybridization (FISH) resulted in a bright signal for the highly amplified sequence in the transfectants, and these signals located at either extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSR), whose appearance was very close to the one that was generated during human malignant transformation. The method is simple, rapid, and highly effective, generating DMs or HSRs bearing thousands of copies of transgenes per human COLO 320 cell in more than 80% of the transfectants within about one month. Presence of both IR and MAR sequences in the plasmid was required for the efficient amplification [6], [7], and deletion of either.
