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

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• 作者：Tomohiro Yoshikawa ; Fumi Nakanishi ; Yuki Ogura ; Daisuke Oi ; Takeshi Omasa ; Yoshio Katakura ; Michimasa Kishimoto ; and Ken-ichi Suga
• 刊名：Biotechnology Progress
• 出版年：2000
• 出版时间：October 2000
• 年：2000
• 卷：16
• 期：5
• 页码：710 - 715
• 全文大小：168K
• 年卷期：v.16,no.5(October 2000)
• ISSN：1520-6033

文摘

Previously, we established an easy and quick construction method for obtaining a stableand highly productive gene-amplified recombinant Chinese hamster ovary (CHO) cellline. With a gradual increase in methotrexate (MTX) concentration, gene-amplifiedcell pools had high and stable specific growth and production rates. Moreover, thephenotype of gene-amplified cells seemed to be affected by the location of the amplifiedgene in chromosomal DNA. We suspected that various kinds of gene-amplified cellsmight 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 MTXconcentration, we isolated gene-amplified clones derived from gene-amplified cell pools.We compared the characteristics of isolated clones, such as the productivity ofrecombinant 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 thetelomeric region, were found to be more stable and productive than other types ofclones. Telomere-type clones had over 100 copies of amplified genes in the chromosomalDNA. In contrast, a large number of other types of clones had less than 10 copies ofamplified genes. During long-term cultivation in the absence of MTX, in other typesof clones, amplified genes rapidly decreased in the chromosomal DNA.