Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, various kinds of stepwise methotrexate (MTX) selection were carried out. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth and production rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage of amplified genes near the telomeric region hardly changed, but that of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productive and stable gene-amplified cells based on the chromosomal location of the amplified gene.