OVEREXPRESSION OF HUMAN PROTHROMBIN IN PERMANENT CELL-LINES USING A DOMINANT SELECTION AMPLIFICATION FUSION MARKER

Human prothrombin was overexpressed in transformed eukaryotic cells us ing a dominant bifunctional selection and amplification marker. The ma rker consists of the murine wild-type dihydrofolate reductase (dhfr) c DNA and the Escherichia coli hygromycin phosphotransferase gene fused in frame, The gene of interest is connected by the encephalomyocarditi s virus 5' untranslated region to the fusion marker gene, forming a di cistronic transcription unit, The human prothrombin gene (FII) was use d to monitor expression after initial selection for hygromycin B resis tance 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 ce lls levels of 20-50 mU/10(6) cells per 24 h were obtained. Methotrexat e-mediated amplification of the foreign gene in CHO cells resulted in a more than 10-fold increase in PII 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 prothromb in were thus obtained. (C) 1996 Academic Press, Inc.