Evaluation Of Stability In The Dhfr Gene Amplification System Using Fluorescence In Situ Hybridization

Tn order to obtain a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, five binds of stepwise methotrexate (MTX) selection were carried out. The specific growth and production rates of the cells were compared with each other, and the distribution of the location of amplified genes was determined using fluorescence in situ hybridization (FISH). The specific growth and production rates of the cell line obtained under the selection condition in which the stepwise increase in the MTX concentration was most gradual reached the highest levels and under this condition about 80% of the amplified genes were observed near the telomeric site. During long-term cultivation without MTX, the percentage of amplified genes near the telomeric site hardly changed, but that of amplified genes at other sites decreased. The specific production rate gradually decreased during cultivation without MTX To clarify the relationship between the specific production rate and the location of amplified genes, a cloned cell line, DR1000L4N, was obtained. This cell line showed higher productivity, and the amplified genes were all situated near the telomeric site.