• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.1
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• pp.31-36
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• 2006

The aim of this study was to elucidate the mechanism of anemia associated with Dangguibohyel-tang (DBT) in rats. Using cyclophosphamide-induced (30mg/kg BW) anemic rats, changes in weight gain, the levels of red blood cell (RBC), hematocrit (Hct), platelet and hemoglobin (Hgb), serum vitamin B12, ALT(GPT) levels and erythropoietin (EPO) gene expression were monitored, and compared with DBT (1,000mg/kg BW, 14d)-treated and EPO (1,000IU/kg BW, 14d, s.c.)-treated rats. Food efficiency ratio (FER) were 31.6%, in normal group, 28.1% in cyclophosphamide-induced control group, 31.7% in DBT-treated group and 25.1%, in EPO-treated group after 14 days. The levels of red blood cell (RBC), hematocrit (Hct), platelet and hemoglobin (Hgb) of DBT-treated group were significantly higher than those of control. And DBT extract administered group showed dominant effects on the recovery of Hgb level. Serum vitamin B12 and ALT(GPT) levels were significantly increased at DBT-treated groups. EPO gene expression was decreased 91,9% in control group, 79.6% in DBT-treated group and 53.9% in EPO-treated group, respectively. These results suggest that administration of DBT could prevent human patient from chemotherapy derived anemia by improving hematological value and EPO status.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.221-224
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• 2001

Effect of hyperosmotic pressure on growth of recombinant Chinese hamster 。 vary cells and Erythropoietin (EPO) production was investigated. Cells were cultivated in batch modes at various osmolalities. When the osmolality increased from 314 to 463mOsm/Kg, specific EPO productivity (qp) was increased up to 1.6-fold but cell growth was inhibited. EPO has a complex oligosaccharide structure that plays an important role in biological activity in vivo. To investigate the influence of hypoerosmotic pressure on the glycosylation, structural analysis of oligosaccharide was calTied out. Recombinant human EPO was produced by CHO cells grown under various osmotic pressure and purified from culture supernatants by heparin-sepharose affinity column and immunoaffinity column. N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were labeled with fluorescent dye, 2-aminobenzamide and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of GU (glucose unit) value. Glycan analysis by HPLC showed that neutral (asialo) oligosaccharide was increased slightly with an increase in osmolality. In portion of sialylated glycan, total relative amount of mono- and di-sialyated glycan was increased but that of tri- and tetra-sialylated glycan decreased as osmolality was increased.

• Biomolecules & Therapeutics
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• v.4 no.4
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• pp.323-329
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• 1996

A recombinant human erythropoietin (rHu-EPO), was administered intravenously to beagle dogs at doses of 100, 500 and 2, 500IU/kg/day for 30 days. There were no significant clinical signs such as body weight, food intake, physical and opthalmic examination, urine analysis, etc. Any toxic response was not observed except for enlarged spleen and extramedullary hematopoiesis. No observed adverse effect level (NOAEL) of rHu-EPO for 30 days was considered to be 100IU/kg/day in beagle dogs.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1342-1351
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• 2008

The feasibility of the use of Flp-mediated cassette exchange in the development of a CHO cell line, which produces erythropoietin (EPO) stably and largely, was investigated. A stable, high enhanced green fluorescence protein (EGFP)-producing clone was screened by extensive flow cytometric analysis. An EPO expression unit was targeted into the premarked locus of the stable parental clone by Flp-mediated cassette exchange and a correctly targeted clone (FC28T7) was obtained. The EPO production of FC28T7 was proven to be stable in long-term culture. Furthermore, the Flp-mediated cassette exchange did not alter the stable parental clone's characteristics concerning transgene expression level and stability. Taken together, the data obtained here indicated that the establishment of CHO cell lines stably producing a desired protein is achievable using Flp-mediated cassette exchange.

• 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 PSK Conference
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• 2003.04a
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• pp.231.2-231.2
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• 2003

The efficient EPO (Erythropoietin) expression system in Chinese Hamster Ovary (CHO) cells was devised through the removal of ammonium ion accumulated in the media by introducing urea cycle enzymes. Previously, we developed C05 cell by transfecting the carbamoly phosphate synthase (CPS) and ornithine transcarbamoylase (OTC) into the EPO expressing CHO cell, IBE. (omitted)

• Applied Microscopy
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• v.37 no.3
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• pp.167-174
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• 2007

Heat shock protein (HSP) 70 functions as a molecular chaperon and reduces stress-induced denaturation and aggregation of intracellular proteins. Erythropoietin (EPO) plays an important role during acute renal failure repair process by rapidly correcting anemia and enhancing renal tubular regeneration. The purpose of this study was to examine the effect of EPO treatment on renal HSP70 expression. Male Sprague-Dawley rats were injected rHUEPO. Kidney were preserved by in vivo perfusion with paraformaldehyde-lysine-periodate (PLP) and processed for immunohistochemistry and electron microscopy. In control kidney, HSP70 was expressed in the cortex, outer medulla and inner medulla. Especially, HSP immunoreactiviy was mainly founded in descending thin limb of outer medulla and inner medullary collecting duct. In EPO treated kidney, HSP70 expression markedly increased in the descending thin limb of outer medulla and newly detected in cortical collecting duct. Electron microscopy showed the presence of HSP immunoreactivity on the intracelluar vesicles and Golgi complex of descending thin limb and cortical collecting duct. These findings suggest that EPO treatment leads to new production of HSP70 in renal tubular cells, and induction of HSP70 by rHuEPO is causally related to protective function.

• 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.

• Biomolecules & Therapeutics
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• v.6 no.1
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• pp.56-62
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• 1998

Mutagenicity of recombinant human erythropoietin (rhEPO) was examined in the reverse mutation test on bacteria, in the chromosomal aberration test on cultured mammalian cells and in the micronucleus test on mice. The reverse mutation test was performed by a plate incorporation method with or wothout a metabolic activation system (59 Mix) using Salmonella typhimurium strain TA100, TA1535, TA98 and TA 1537. The rhEPO did not significantly increase revertant colonies in any of the test strains under any conditions at dose levels ranging from 1000 H/ml to 62.5 lu/plate, compared with the vehicle control. In the chromosomal aberration test using cultured Chinese Hamster Lung (CHL) cells, the number of aberrant cells was not increased in the presence or absence of 59 Mix at concentrations of 1000 lU/ml to 250 lU/ml, compared with the vehicle control. In the micronucleus test, male ICR mice were given rhEPO intraperitoneally at a dose level of 25000, 12500 and 6250 lU/kg. The incidence of bone marrow micronucleated polychromatic erythrocytes was not different from that of the vehicle control. From these results, rhEPO is considered to be non-mutagenic under the present test conditions.

• Molecules and Cells
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• v.23 no.1
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• pp.17-22
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• 2007

We have expressed human erythropoietin (EPO) in transgenic mice using a recombinant EPO cDNA combined with a partial TPO construct. The gene was microinjected using standard techniques and five mice were detected as transgenic by PCR and further used as founders. The life span of the transgenic founders was much shorter than that of their normal littermates. Most of the tissues of the transgenic founders contained human EPO transcripts as judged by RT-PCR. Especially high expression levels were seen in the liver and lung. EPO protein levels in serum were examined by ELISA and ranged from 266-414 mIU/ml. The number of red blood cell, white blood cell and hemoglobin in the hEPO transgenic mice was higher than in normal mice. These results indicate that overexpression of hEPO is deleterious and can provoke lung failure and erythrocytosis.