Identification and analysis of specific chromosomal region adjacent to exogenous Dhfr-amplified region in Chinese hamster ovary cell genome

Chinese hamster ovary (CHO) cells are widely used for the stable production of recombinant proteins. Gene amplification techniques are frequently used to improve of protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used in 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 one BAC clone (Cg0031N14) containing the CHO genomic DNA sequence adjacent to Dhfr was selected. To identify the specific chromosomal region adjacent to the exogenous Dhfr-amplified region in the CHO cell genome, we performed further screening of BAC clones to obtain other Dhfr-amplified regions in the CHO genome. From the screening by high-density replica filter hybridization using a digoxigenin-labeled pSV2-dhfr/hGM-CSF probe, we obtained 8 new BAC clones containing a Dhfr-amplified region. To define the structures of the 8 BAC clones, Southern blot analysis, BAC end sequencing and fluorescence in situ hybridization (FISH) were performed. These results revealed that all the selected BAC clones contained a large palindrome structure with a small inverted repeat in the junction region. This suggests that the obtained amplicon structure in the Dhfr-amplified region in the CHO genome plays an important role in exogenous gene amplification.