• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.193-199
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• 2010

We report here the production of transgenic chickens that can regulate human erythropoietin (hEPO) gene expression. The glycoprotein hormone hEPO is an essential for viability and growth of the erythrocytic progenitors. Retrovirus vector system used in this study has two features including tetracycline-controllable promoter and woodchuck hepatitis virus posttranscriptional regulator element (WPRE). The former is for to reduce the possibility of physiological disturbance due to constitutional and unregulated expression of hEPO gene in the transgenic chicken. The latter is for maximum expression of the foreign gene when we turn-on the gene expression. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 325 injected eggs, 28 chicks hatched after 21 days of incubation and 16 hatched chicks were found to express the hEPO gene delivered by the vector. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. The recombinant hEPO in transgenic chicken serum had N- and O-linked carbohydrate simillar to that produced from in vitro cultured cells transformed with hEPO gene.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.17-17
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• 2003

The milk of transgenic animals may provide an attractive vehicle for large-scale production of hEPO. Since glycosylation is cell type specific, recombinant human EPO (rhEPO) produced in different host cells contain different patterns of oligosaccharides, which could affect the biological functions. However, there have been no reports on the characteristics of rhEPO derived from milk of transgenic animals. To address this objective, several transgenic mice by using pWAPhEPO and/or pBC1hEPO expression vector were produced. However, 2 lines of pWAPhEPO founder female mouse died during late gestational day (day 18) before offspring could be obtained. They showed a severe splenomegaly, Unlike those of pWAPhEPO, mammary gland epithelial cells from biopsies of lactating pBC1hEPO transgenic mice had marked immunoreactivity to EPO and any activity was not detected in other tissues. The expression level of rhEPO is about 0.7% of mammary gland cellular total soluble proteins and an amount of 300~500 mg/L rhEPO is secreted into milk. Furthermore, the pBC1hEPO transgenic mice transmitted this character to their progeny in mendelian manner. In order to determine the extent of glycosylation variation, N-linked oligosaccharide structures present in the milk-derived rhEPO were characterized. Most of milk-derived rhEPO is fully glycosylated. the biological activity of milk-derived rhEPO was comparable to that of purified CHO-derived rhEPO, and milk-derived rhEPO showed relatively stable after freezing and thawing. Taken together, the results illustrate the potential of transgenic animals in the large-scale production of biopharmaceuticals.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.77-83
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• 2009

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. We constructed three EPO mutants ($\Delta$69, $\Delta$105 and $\Delta$69,105), containing an additional oligosaccharide chains. EPOWT and EPO$\Delta$69 were effectively expressed in transient and stably transfected CHO-K1 cell lines. But, it wasn't detected any protein in the culture medium of EPO$\Delta$105 and EPO$\Delta$69,105 mutants. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure the cytokine dependency and in vitro bioactivity of rec-hEPO. MTT assay values were increased by survival of F36E cells at 24h. To analysis biological activity in vivo, two groups of ICR-mice (7 weeks old) were injected subcutaneously with 10 IU per mice of rec-hEPO molecules on days 0 and 2. Red blood cell and hematocrit values were measured on 6 days after the first injection. The hematocrit values were remarkably increased in all treatment groups. The pharmacokinetic analysis was also affected in the mice injected with rec-hEPO molecules 2.5 IU by tail intravenous. Protein samples were detected by Western blotting. An EPO$\Delta$69 protein migrated as a broad band with an average apparent molecular and detected slightly high band. Enzymatic N-deglycosylation resulted in narrow band and was the same molecular size. The biological activity of EPO$\Delta$69 was enhanced to compare with wt-hEPO. The half-life was longer than wt-hEPO. The results suggest that hyperglycosyalted recombinant human erythropoietin (EPO$\Delta$69) may have important biological and therapeutic good points.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.60-60
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• 2002

본 연구는 사람의 조혈촉진유전자가 형질전환된 돼지의 유즙내 hEPO 발현농도를 확인하고 3종의 동물 (mouse, rat, swine)에서 hEPO 의 생리활성을 분석하기 위하여 실시하였다. 새롬이 Fl 암컷의 유즙을 원심분리하여 지방을 제거한 후 hEPO 의 농도를 측정하고, PBS 로 유즙내 hEPO 농도가 100 IU/㎖가 되도록 희석하였다. Mouse (ICR)는 피하주사로 20 IU씩 2일 간격으로 3회 (0, 2, 4 일차 ) 주사하였으며, 주사 후 4회에 걸쳐 (0, 2, 4, 7 일차) 혈액을 채취하여. 분석하였다. (중략)

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.69-73
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• 2005

The hematopoietic growth factor erythropoietin (EPO) is required for the maintenance, proliferation, and differentiation of the stem cells that produce erythrocytes. To analyse the biological activity of the recombinant human EPO (rec-hEPO), we have cloned the EPO cDNA and genomic DNA and produced rec-hEPO in the CHO cell lines. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of rec-hEPO. MIT assay values were increased by survival of F36E cells at 24h or 72h. The hematocrit and RBC values were increased by subcutaneous injection of 20 IU (in mice) and 100IU(in rats) rec-hEPO. Hematocrit values remarkably increased at $13.2\%$ (in mice) and $12.2\%$ (in rats). The pharmacokinetic behavior with injection of 6 IU of rec-hEPO remained detectable after 24 h in all mice tested. The highest peat appeared at 2h after injection. The long half-life of rec-hEPO is likely to confer clinical advantages by allowing less frequent dosing in patients treated for anemia. These data demonstratethat ree-hEPO produced in this study has a potent activity in vivo and in vitro. The results also suggest that biological activity of ree-hEPO could be remarkably enhanced by genetic engineering that affects the potential activity, including mutants with added oligosaccharide chain and designed to produce EPO-EPO fusion protein.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.21-21
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• 2002

사람의 erythropoietin (hEPO) 는 산성 당단백질 호르몬이며 적혈구 생산의 주요조절인자로서 적혈구의 분화와 hemoglobin (Hb) 형성을 촉진하여 빈혈치료제로 이용된다. 사람 EPO 는 166개 아미노산으로 구성되어 있으며, 24, 38, 83 번 아미노산은 N-glycosylation에 의해, 126 번 아미노산은 O-glycosylation에 의해 변형되며, 특히 N-glycosylation은 hEPO 의 세포외 분비 및 활성에 관여한다고 보고된 바 있다. (중략)

• Neonatal Medicine
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• v.16 no.2
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• pp.221-233
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• 2009

Purpose: Erythropoietin (EPO) has neuroprotective effects in many animal models of brain injury, including hypoxic-ischemic (HI) encephalopathy, trauma, and excitotoxicity. Current studies have demonstrated the neuroprotective effects of EPO, but limited data are available for the neonatal periods. Here in we investigated whether recombinant human EPO (rHuEPO) can protect the developing rat brain from HI injury via modulation of NMDA receptors. Methods: In an in vitro model, embryonic cortical neuronal cell cultures from Sprague-Dawley (SD) rats at 19-days gestation were established. The cultured cells were divided into five groups: normoxia (N), hypoxia (H), and 1, 10, and 100 IU/mL rHuEPO-treated (H+E1, H+ E10, and H+E100) groups. To estimate cell viability and growth, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was done. In an in vivo model, left carotid artery ligation was performed on 7-day-old SD rat pups. The animals were divided into six groups; normoxia control (NC), normoxia Sham-operated (NS), hypoxia-ischemia only (H), hypoxia-ischemia+vehicle (HV), hypoxia-ischemia+rHuEPO before a HI injury (HE-B), and hypoxia-ischemia+rHuEPO after a HI injury (HE-A). The morphologic changes following brain injuries were noted using hematoxylin and eosin (H/E) staining. Real-time PCR using primers of subunits of NMDA receptors (NR1, NR2A, NR2B, NR2C and NR2D) mRNA were performed. Results: Cell viability in the H group was decreased to less than 60% of that in the N group. In the H+E1 and H+E10 groups, cell viability was increased to >80% of the N group, but cell viability in the H+E100 group did not recover. The percentage of the left hemisphere area compared the to the right hemisphere area were 98.9% in the NC group, 99.1% in the NS group, 57.1% in the H group, 57.0% in the HV group, 87.6% in the HE-B group, and 91.6% in the HE-A group. Real-time PCR analysis of the expressions of subunits of NMDA receptors mRNAs in the in vitro and in vivo neonatal HI brain injuries generally revealed that the expression in the H group was decreased compared to the N group and the expressions in the rHuEPO-treated groups was increased compared to the H group. Conclusion: rHuEPO has neuroprotective property in perinatal HI brain injury via modulation of N-methyl-D-aspartate receptors.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.317-324
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• 2003

The present study were performed to analysis the hematocrit and the red blood cells content into the blood plasma of the transgenic pigs harboring recombinent human erythropoietin gene (rhEPO). Mouse whey acidic protein (mWAP) linked to rhEPO gene was microinjected into pronuclei of porcine one-cell zygotes. After delivered of offspring, PCR analyses identified one mWAP-rhEPO transgenic founder offspring(F$_{0}$). The first generation of transgenic pig (F$_{0}$) harboring mWAP-hEPO appeared to be a male, and the second generation (F$_1$) pigs were made by natural mating of F$_{0}$ with domestic swine, and male and female transgenic pigs (F$_1$) were identified by PCR. The blood samples from transgenic and normal pigs were collected for 50 days during lactation and were counted the red blood cell (RBC) numbers and Hematocrit (HCT) content into the blood. The transgenic pigs expressing rhEPO in their blood gave rise to higher RBC numbers and HCT contents than control animals. rhEPO was secreted both in the blood and milk of genetically engineered pigs harboring rhEPO gene. Therefore, this study provides a model regarding the production of transgenic pig carrying hEPO transgene for biomedical research.earch.

• Bulletin of the Korean Chemical Society
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• v.34 no.9
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• pp.2663-2670
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• 2013

The purpose of stability testing is to provide evidence about how the quality of a drug varies with time under the influence of a variety of environmental factors. In this study, erythropoietin (EPO) was analyzed under different pH (pH 3 and pH 9) and temperature ($25^{\circ}C$ and $40^{\circ}C$) conditions according to current Good Manufacturing Practice (cGMP) and International Conference on Harmonisation (ICH) guidelines. The molecular weight difference between intact EPO and deglycosylated EPO was determined by SDS-PAGE, and aggregated forms of EPO under thermal stress and high-pH conditions were investigated by size exclusion chromatography. High pH and high temperature induced increases in dimer and high molecular weight aggregate forms of EPO. UPLC-ESI-TOF-MS was applied to analyze the changed modification sites on EPO. Further, normal-phase high-performance liquid chromatography was performed to identify proposed glycan structures and high pH anion exchange chromatography was carried out to investigate any change in carbohydrate composition. The results demonstrated that there were no changes in modification sites or the glycan structure under severe conditions; however, the number of dimers and aggregates increased at $40^{\circ}C$ and pH 9, respectively.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.582-586
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• 2003

There has been interest in developing gene therapy based on naked plasmid DNA for treating serum protein deficiencies and human erythropoietin (hEPO) is one of the candidate for gene therapy being Investigated most enthusiastically. We constructed novel plasmid DNA vectors pVAC-hEPOI/II/III which contain one, two, three hEPO gene(s) respectively for producing high level expression and secretion of hEPO in vitro and in vivo. NIH3T3 and COS7 cells were transfected transiently with these vectors and increase in hEPO expression in medium reached 2-5 fold in comparison with pSecTagB-hEPO. Intra muscular administrations of pVAC-hEPOI/II/III vectors into mice resulted in high level secretion of hEPO in the serum and corresponding increases in hematocrit level. In conclusion the transduction efficiency of naked plasmid vectors is one of the critical factors of a gene delivery system and these novel plasmid vectors will contribute to various gene therapy based on naked plasmid DNA.