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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.
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Expression and Functional Characterization of Recombinant Human Erythropoietin (rhEPO) Produced in the Milk of Transgenic Mice
한국동물번식학회 학술대회
2003 .01
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Effects of Mutagenesis for Glycosylation Sites of Recombinant Human EPO During Production from Cultured CHO Cell
한국수정란이식학회 심포지움
2002 .01
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Development and Characterization of Hyperglycosylated Recombinant Human Erythropoietin (HGEPO)
한국동물번식학회지
2009 .01
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Biological Activity of Recombinant Human Erythropoietin (EPO) In Vivo and In Vitro
한국동물번식학회지
2005 .01
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Characterizations of Cell Lineage Markers in the Bone Marrow Cells of Recloned GFP Pigs for Possible Use of Stem Cell Population
한국동물번식학회지
2011 .01
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Double Reporter Genes Enabling the Viable and Reliable Selection, and cell Tracing
한국수정란이식학회 심포지움
2005 .01
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hEPO 당쇄부위 돌연변이체 제작 및 CHO 세포로부터 변이 단백질의 생산
한국동물번식학회 학술대회
2002 .01
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Increased of the Red Blood Cell in Peripheral Plasma of Transgenic Pigs Harboring hEPO Gene
한국가축번식학회지
2003 .01
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Biological roles and activities of N - linked glycosylation site addition at $Asn^{69}$ of Dimeric EPO
한국수정란이식학회 심포지움
2005 .01
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닭고기의 감마지방산 강화에 관한 달맞이꽃종자유의 급여효과
한국식품영양과학회지
2007 .06
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소변으로 EPO를 분비하는 형질전환 돼지생산
한국동물번식학회 학술대회
2001 .01
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Use of Animal Viral Internal Ribosome Entry Site Sequence Makes Multiple Truncated Transcripts without Mediating Polycistronic Expression in Rice
Applied Biological Chemistry
2011 .01
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Biological Activity of Human Dimeric Hyperglycosylated Erythropoietin (dHGEPO) Fusion Proteins
한국동물번식학회지
2010 .01
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Efficient Expression of hG-CSF cDNA from an IRES-Dependent Bicistronic Vector Targeted to Mammary Gland of Transgenic Mice
한국동물번식학회 학술대회
2002 .01
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GFP Gene Transfected Cell 과 Non Tranfsfected Cell 의 핵이식후 발달
한국동물번식학회 학술대회
2002 .01
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