Efficient recombinant production in mammalian cells using a novel IR/MAR gene amplification method

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 establishme...

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Bibliographic Details
Published in:PloS one 2012-07, Vol.7 (7), p.e41787-e41787
Main Authors: Araki, Yoshio, Hamafuji, Tetsuro, Noguchi, Chiemi, Shimizu, Noriaki
Format: Article
Language:English
Subjects:
Amplification
Analysis
Animals
Antibodies
Biology
Biosphere
Cell culture
Cell cycle
Cell Line, Tumor
Cells (Biology)
CHO Cells
Cricetinae
Cricetulus
Deoxyribonucleic acid
DNA
DNA methylation
DNA Replication - genetics
DNA, Recombinant - biosynthesis
DNA, Recombinant - genetics
Gene amplification
Gene expression
Genes
Genetic Engineering - methods
Genetic research
Genomes
Humans
Immunoglobulin G - biosynthesis
Immunoglobulin G - genetics
Mammalian cells
Mammals
Matrix Attachment Regions - genetics
Methods
Nucleic Acid Amplification Techniques - methods
Plasmids
Plasmids - genetics
Productivity
Proteins
Replication initiation
Rodents
Suspension culture
Transfection
Yeast
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0041787