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.
Highlights
Amplification of oncogenes or drug-resistance genes plays a pivotal role in malignant transformation of human cells via over-production of the corresponding protein products; understanding the gene amplification process has important implications for cancer research
We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected cells, and efficiently generate chromosomal homogeneously staining regions (HSRs) and/or extrachromosomal double minutes (DMs) [13,14], which are cytogenetic manifestations of gene amplification
Chinese hamster ovary (CHO) DXB11 cells were transfected with the IR/MARpositive pDBM AR1Dhfr-Fc receptor gene (FcR) plasmid (Figure 1A) or the IR/ MAR-negative pECE-rshFcR dihydrofolate reductase gene (Dhfr) plasmid [23]
Summary
Amplification of oncogenes or drug-resistance genes plays a pivotal role in malignant transformation of human cells via over-production of the corresponding protein products; understanding the gene amplification process has important implications for cancer research (reviewed in [1,2,3]). The clone showing the highest level of FcR production obtained by each method was cultured in increasing concentrations of Mtx. Compared with the cultures produced by the conventional Dhfr/ Mtx method, FcR expression increased much more rapidly in the IR/MAR-Dhfr fusion cultures (Figure 1); detection of the plasmid sequence by FISH suggested that this amplification progressed rapidly and dramatically (data not shown).
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