Overexpression of human prothrombin in permanent cell lines using a dominant selection/amplification fusion marker.

Human prothrombin was overexpressed in transformed eukaryotic cells using a dominant bifunctional selection and amplification marker. The marker consists of the murine wild-type dihydrofolate reductase (dhfr) cDNA and the Escherichia coli hygromycin phosphotransferase gene fused in frame. The gene of interest is connected by the encephalomyocarditis virus 5' untranslated region to the fusion marker gene, forming a dicistronic transcription unit. The human prothrombin gene (FII) was used to monitor expression after initial selection for hygromycin B resistance and DHFR activity. In Chinese hamster ovary (CHO) cells, 5-15 mU prothrombin/10(6) cells per 24 h was obtained; in human 293 kidney cells levels of 20-50 mU/ 10(6) cells per 24 h were obtained. Methotrexate-mediated amplification of the foreign gene in CHO cells resulted in a more than 10-fold increase in FII expression, while in the presence of methotrexate, 293 cells expressed 200-250 mU/10(6) cells per 24 h. The use of this fusion marker within a dicistronic transcription unit allowed efficient dominant selection of cell clones and amplification of the gene of interest. Stably transfected cell lines that were able to secrete high levels of processed gamma-carboxylated human prothrombin were thus obtained.