Chinese hamster ovary (CHO) cells are widely used in producing therapeutic proteins. Gene amplification techniques are frequently used in improving protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used for the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and found one BAC clone (Cg0031N14) containing a CHO genomic DNA sequence adjacent to Dhfr. The BAC clone contained a large palindrome structure with a small inverted repeat in the junction region. To investigate the effect of the palindrome structure derived from the BAC clone Cg0031N14 on Dhfr amplification in CHO cells, we constructed plasmids that contain part or the whole junction region of the palindrome structure. The transfected CHO DG44 cells containing part or the whole junction region of the palindrome structure could adapt quickly to high methotrexate (MTX) concentrations. Moreover, the cells containing the whole junction region of the palindrome structure showed a high ratio of GFP-positive cells during gene amplification. On the basis of these results, we estimated that the junction region plays an important role in gene amplification in CHO cells. (C) 2009 Elsevier Ltd. All rights reserved.