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Effect of IRES Controlled Reporter Gene on Screening and Production of Recombinant Human EPO Proteins from Cultured CHO Cells  

Lee Hyun Gi (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Park Jin-Ki (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Kim Sung-Woo (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Ko Eun-Mi (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Kim Byoung-Ju (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Jo Su-Jin (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Byun Sung-June (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Yang Boh-Suk (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Chang Won-Kyong (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Lee Hoon-Taek (Department of Animal Biotechnology, KonKuk University)
Lee Poong-Yeon (Transgenic Research Lab., Animal Biotechnology Division, NLRl, RDA)
Publication Information
Reproductive and Developmental Biology / v.30, no.2, 2006 , pp. 81-85 More about this Journal
Abstract
This study was conducted to examine the effect of IRES controlled reporter gene on screening and production of recombinant human erythropoietin (EPO) proteins from cultured CHO cells. The cDNA was cloned for EPO from human liver cDNA Using site-directed mutagenesis, we generated recombinant human EPO (rhEPO) with two additional N-glycosylations (Novel erythropoiesis-stimulating protein: NESP). Wild type hEPO and NESP were cloned into expression vectors with GFP reporter gene under regulatory control of CMV promoter and IRES so that the vectors could express both rhEPO and GFP. The expression vectors were transfected to cultured CHO-K1 cells. Under microscopy, expression of GFP was visible. Using supernatant of the culture, ELISA assay, immunocytochemistry and in vitro assay using EPO dependant cell line were performed to estimate biological activity to compare the production characteristics (secretion levels, etc.) between rhEPO and NESP. The activity of NESP protein, obtained by mutagenesis, was described and compared with its rhEPO counterpart produced under same conditions. Although NESP had less secretion level in CHO cell line, the biological activity of NESP was greater than that of rhEPO. These results are consistent with previous researches. We also demonstrated that rhEPO and GFP proteins expressed simultaneously from transfected CHO cell line. Therefore we conclude that use of GFP reporter gene under IRES control could be used to screen and produce rhEPO in cultured CHO cells.
Keywords
hEPO; Glycosylation site; Site-directed mutagenesis; IRES; GFP; CHO cell;
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