Co - Transfection of EPO, GFP and DHFR genes into mammalian cells and selection of cells with highly and stably expressed erythropoietin

Abstract

Recombinant human erythropoietin (rHuEpo) has been used widely to treat anemia in patients with chronic renal failure. Its production requires a high and stable expression system with mammalian cell as a host. In this study the lipofectamine using co-transfection of three plasmids pCDNAEPO1 sequence as GOI with pSV2-dhfr and pEGFP-N1 as selectable markers was carried out as a developing strategy to improve the selection and productivity of CHO-dhfr(-) cells with stable expression of erythropoietin. The total transfected DNA amount was 1 µg and the suitable gene ratio gfp:epo was 1:3 and dhfr: (gfp+epo) was 1:5. The transfectants were transiently evaluated about EPO expression by Dot-blot test and then selected in the 100µg/mL zeocin - HT minus CHO-DHFR(-) selective medium. The selected cells were quantitatively evaluated about stable EPO expression by indirect ELISA and cloned by the GFP-based Fluorescence Activated Cell Sorting (FACS) system. As a result, a highly and stably EPO producing cell line denoted S4 cell line was created with the EPO productivity 2.468 pg/cell/day and possessed dhfr gene as a preparation for further dhfrbased gene amplification of EPO production in next study. Key words: Recombinant human erythropoietin (rHuEpo), CHO-dhfr(-) cell, cotransfection, EPO, DHFR, GFP, stable expression, EPO productivity, FACS.