Effects of palindrome structure on Dhfr amplification in Chinese hamster ovary cells |
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| Affiliation: | 1. Department of Medicinal and Pharmaceutical Chemistry, Korea University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-333, Republic of Korea;2. Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong-gu, Daejeon 305-600, Republic of Korea;2. AB Vista Feed Ingredients, Marlborough, Wiltshire, SN8 4AN, United Kingdom;1. Department of Animal Biotechnology, KonKuk University, Seoul, Republic of Korea;2. Animal Biotechnology Division, National Institute of Animal Science, RDA, Gyeonggi-do, Republic of Korea;1. Institute of Science and Innovation in Mechanical and Industrial Engineering (INEGI), University of Porto, Campus da FEUP, Av. Roberto Frias, 400, 4200-465 Porto, Portugal;2. LAETA, IDMEC, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal;3. Faculdade de Engenharia da Universidade do Porto, Campus da FEUP, Av. Roberto Frias, s/n, 4200-465 Porto, Portugal;1. Key Laboratory of Theoretical Chemistry of Environment, Ministry of Education, School of Chemistry and Environment, South China Normal University, Guangzhou 510006, PR China;2. School of Chemistry and Environment, South China Normal University, Guangzhou 510006, PR China;3. Guangzhou Key Laboratory of Materials for Energy Conversion and Storage, Guangzhou 510006, PR China |
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| Abstract: | 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. |
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