Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes

Previously, we established an easy and quick construction method for obtain ing a stable and highly productive gene-amplified recombinant Chinese hamst er ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) con centration, gene-amplified cell pools had high and stable specific growth a nd production rates. Moreover, the phenotype of gene-amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. W e suspected that various kinds of gene-amplified cells might appear during the long-term selection to construct gene-amplified cell pools. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, me isolated gene-amplified clones derived from gene-amplifi ed cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere-type clones, in whic h the amplified gene was located near the telomeric region, were found to b e more stable and productive than other types of clones. Telomere-type clon es had over 100 copies of amplified genes in the chromosomal DNA. In contra st, a large number of other types of clones had less than 10 copies of ampl ified genes. During long-term cultivation in the absence of MTX, in other t ypes of clones, amplified genes rapidly decreased in the chromosomal DNA.