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Biotechnology progress

Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes.


PMID 11027161

Abstract

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene-amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene-amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene-amplified cells might appear during the long-term selection to construct gene-amplified cell pools. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, we isolated gene-amplified clones derived from gene-amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.

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