Efficiency of promoter and cell line in high‐level expression of erythropoietin

Efficiency of viral promoters and various cell lines in directing high-level expression of human erythropoietin (Epo) was investigated. To investigate the effects of various viral promoters and cell lines on the Epo expression level, genomic Epo with the 5' and 3' untranslated regions (UTRs) deleted was cloned next to the simian virus 40 early promoter, cytomegalovirus early promoter or SRalpha promoter. These expression vectors were transfected into COS-7, BHK-21 and Chinese hamster ovary (CHO)/dhfr(-) cells, respectively. The COS-7 cells transfected with the vector containing the SRalpha promoter showed the highest expression level ( approximately 103 IU/ml) at 72 h post-transfection. For the development of Epo-producing stable cell lines, BHK-21 and CHO/dhfr(-) cells transfected with the 5',3'-UTR-deleted genomic Epo under the control of the SRalpha promoter were cultured with media containing zeocin. Several clones of zeocin-resistant BHK-21 and CHO/dhfr(-) cells were cultured in the presence of methotrexate (MTX). A BHK-21 clone selected in the presence of 500 nM MTX expressed and secreted approximately 490 IU/ml Epo into the medium. A CHO/dhfr(-) clone selected in the presence of 20 nM MTX expressed and secreted approximately 45 IU/ml Epo into the medium. Southern-blot analysis indicated that enhancement of Epo expression in the MTX-resistant stable cells might be related to the amplification of gene copy number.