Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes.
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.
Yoshikawa
T
T
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita 565-0871, Japan.
Nakanishi
F
F
Ogura
Y
Y
Oi
D
D
Omasa
T
T
Katakura
Y
Y
Kishimoto
M
M
Suga
K
K
D000818:Animals; D016466:CHO Cells; D002875:Chromosomes; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D005784:Gene Amplification; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D008727:Methotrexate / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate