Amplified Gene Location in Chromosomal DNA Affected Recombinant Protein Production and Stability of Amplified Genes

作者：Tomoko Yoshikawa, Fumihiko Nakanishi, Y Ogura, David H. Oi, Takeshi Ōmasa, Yoshio Katakura, Michimasa Kishimoto, Kenichi Suga · 发表于：Biotechnology Progress · 年份：2000 · DOI：10.1021/bp000114e · 被引用次数：95 · 研究领域：Viral Infectious Diseases and Gene Expression in Insects、CRISPR and Genetic Engineering、Virus-based gene therapy research

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.