Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.
Cytotechnology. 2000 Jul;33(1-3):37-46. doi: 10.1023/A:1008111328771.
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, variouskinds of stepwise methotrexate (MTX) selection were carriedout. 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 andproduction 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 ofamplified genes near the telomeric region hardly changed, butthat 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 productiveand stable gene-amplified cells based on the chromosomal location of the amplified gene.
为了建立一种简便快捷的方法来获得稳定高产的基因扩增重组中国仓鼠卵巢（CHO）细胞系，我们进行了各种逐步甲氨蝶呤（MTX）选择。比较了细胞的比生长速率和比生产速率，并通过荧光原位杂交（FISH）确定了扩增基因的位置分布。在 MTX 浓度逐步增加最平缓的选择条件下，细胞库的比生长速率和比生产速率达到最高水平；大约 82%的扩增基因观察到位于端粒区域附近。在没有 MTX 的长期培养过程中，端粒区域附近的扩增基因比例几乎没有变化，但其他区域的扩增基因比例下降。基于这些结果，可以轻松快速地构建稳定高产的细胞库，并进行逐步增加 MTX 浓度的扩增。此外，FISH 技术是一种强大的工具，可根据扩增基因的染色体位置来评估高产稳定的基因扩增细胞。
