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

In previous work, we clarified the relationship between the productivity an d 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 ge nes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed i n other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fl uorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cyt ometry, 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 p roductive gene-amplified cells could be isolated from heterogeneous gene-am plified cell pools more easily than by the method of limiting-dilution assa y. The limiting-dilution method requires several months to obtain highly pr oductive gene-amplified cells, while our flow-cytometry-based method of sel ection requires only a few weeks. (C) 2001 John Wiley & Sons, Inc.