Flow cytometry: An improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry

• Published in: Biotechnology and bioengineering 2001-09, Vol.74 (5), p.435-442
• Main Authors: Yoshikawa, Tomohiro, Nakanishi, Fumi, Ogura, Yuki, Oi, Daisuke, Omasa, Takeshi, Katakura, Yoshio, Kishimoto, Michimasa, Suga, Ken-Ichi
• Format: Article
• Language: English
• Subjects: Animals; Antigens, Surface - analysis; Biological and medical sciences; Biotechnology; Cell Separation - methods; Chinese hamster ovary (CHO); CHO Cells; Cricetinae; dihydrofolate reductase (dhfr); Flow Cytometry - methods; flow-cytometry; fluorescein isothiocyanate; fluorescein-methotrexate (F-MTX); Fluoresceins - analysis; Fundamental and applied biological sciences. Psychology; gene amplification; Gene Amplification - genetics; Genetic engineering; Genetic technics; Granulocyte-Macrophage Colony-Stimulating Factor - analysis; Granulocyte-Macrophage Colony-Stimulating Factor - genetics; Humans; In Situ Hybridization, Fluorescence - methods; Methods. Procedures. Technologies; methotrexate; Methotrexate - analogs & derivatives; Methotrexate - analysis; Miscellaneous; Synthetic digonucleotides and genes. Sequencing; Tetrahydrofolate Dehydrogenase - analysis; Tetrahydrofolate Dehydrogenase - genetics
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.1134

In previous work, we clarified the relationship between the productivity and stability of gene‐amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the “telomere type.” The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the “other type.” The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate‐labeled methotrexate (F‐MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene‐amplified cells could be isolated from heterogeneous gene‐amplified cell pools more easily than by the method of limiting‐dilution assay. The limiting‐dilution method requires several months to obtain highly productive gene‐amplified cells, while our flow‐cytometry‐based method of selection requires only a few weeks. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 435–442, 2001.