Novel cloning strategy to select highly expressed erythropoietin using co-transfection with green fluorescent protein gene

Abstract

Erythropoietin (EPO) is a haemopoietic hormone. It stimulates bone marrow to produce red blood cells in mammals. EPO was used therapeutically for the treatment of anemia associated with chronic kidney failure and the treatment of cancer. In order to increase the productivity of EPO, the high expressed EPO is need as raw material. Therefore, the co-transfection method was used to transfer pEPO and pGFP (green fluorescent protein plasmid) to CHO - K1 cells by lipofectamine kit. The suitable ratio pGFP to pEPO for transfection is 1:3 in 1µg of total DNA. After being detected transient protein expression, those cells were cultured on the selective medium with 100 microgram zeocin per milliliter in two weeks so that cells containing entire vector integrated into chromosomal DNA could survive. Then basing on the expression of GFP, the high expression EPO cells were separated by Fluorescence Activated Cell Sorting (FACS). Finally, the EPO production of stable transfected cells was quantified by indirect ELISA before and after FACS. The amount of EPO produced was increasing dramatically before and after FACS (from 8.1 to 20.4 pg/cell/day at the first time of FACS and to 24.6 at the second time of

FACS). The separated cells may be used for optimization EPO production then applying as raw material for industrially EPO production. Key words: CHO-K1 cell, EPO, GFP, co-transfection, recombinant protein, Fluorescence Activated Cell Sorting (FACS)