• 대한임상약리학회:학술대회논문집
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• 대한임상약리학회 1998년도 제7차 추계학술대회 및 정기총회
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• pp.40-40
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• 1998

• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2001년도 Korean Environmental Mutagen Society
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• pp.168-168
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• 2001

• 한국가축번식학회지
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• 제27권4호
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• pp.299-307
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• 2003

The effects of additions/deletions in glycosylated residues of recombinant human EPO (rhEPO) produced in CHO-K1 on their secretion were examined. hEPO cDNA was amplified from human liver mRNA and cloned into the pCR2.1 TOPO. Using overlapping-extension site-directed mutagenesis method, glycosylation sites at 24th, 38th, 83rd, and 126th were respectively or accumulatively removed by substituting its asparagine (or serine) with glutamine. To add novel glycosylation sites, 69 and 105th leucine was mutated to asparagine. Mutant and wild type rhEPO constructs were cloned into the pcDNA3 expression vector with CMV promoter and transfected into CHO cell line, CHO-K1, to produce mutant rhEPO mutant rhEPO proteins. Enzyme-linked immunosorbant assay (ELISA) and Western analysis with monoclonal anti-EPO antibody were performed using supernatants of the cultures showing transient and stable expressions respectively. Addition of novel glycosylation reduced rhEPO secretion dramatically while deletion mutants had little effect except some double deletion mutants ($\Delta$24/83 and $\Delta$38/83) and triple mutant ($\Delta$24/38/83). This fact suggests that not single but combination of changes in glycosyl groups affect secretion of rhEPO in cell culture, possibly via changes in their conformations.

• Toxicological Research
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• 제21권3호
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• pp.241-247
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• 2005

HM10760 is a recombinant human erythropoietin that has beer developed as a drug for anemia. In this study, antigenic potential of HM10760 was examined by active systemic anaphylaxis in guinea pigs and passive cutaneous anaphylaxis in a guinea pig-guinea pig system. HM10760 was subcutaneously administered at 0, 2, and $20{\mu}g/kg$ and also as a suspension with adjuvant ($20{\mu}g/kg$ + FCA). Ovalbumin as a suspension with adjuvant was administered to induce positive control responses. In the active systemic anaphylaxis test, no symptoms except rubbing or licking nose and urination that were considered as physiological phenomena were observed at $0{\mu}g/kg$. Four of 5 animals at $2{\mu}g/kg$ and all the 5 animals at $20{\mu}g/kg$ showed cyanosis and lying on side. All animals in the adjuvant mixture group showed relatively mild symptoms such as rubbing or licking nose, urination, and evacuation. In the passive cutaneous anaphylaxis test, 0/5, 3/5, and 5/5 serum samples from the animals immunized with 0, 2, and $20{\mu}g/kg$, respectively, showed positive reactions against HM10760. All 5 sera collected from the animals immunized with an adjuvant mixture contained HM10760-specific antibodies. These results suggest HM10760 have antigenicity in guinea pigs.

• Reproductive and Developmental Biology
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• 제30권2호
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• pp.81-85
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• 2006

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.

• Reproductive and Developmental Biology
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• 제40권1호
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• pp.7-13
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• 2016

Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat ${\beta}$-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.

• Toxicological Research
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• 제12권2호
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• pp.309-318
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• 1996

This study was conducted to investigate the subacute intravenous toxicity of rHu-EPO in Sprague-Dawley rats. rHu-EPO was daily administered to male and female rats for 30 days with different dose levels(100, 500, 2,500 unit/ kg). After the administration period of 30 days, 5 males and 5females rats per each dose group were assigned for recovery period of additional 30 days. There were no clinical signs compared with control group but slight decrease in spontaneous motor activities and locomotions were observed at high dose groups of males and females. In males, feed consumption was reduced at 500 unit/kg and body weight gain was retarded. In the administration sites of tail, focal congestion was observed in a few animals treated with rHu-EPO. No deaths were occurred during the course of study. In hematological examination. a significant increase of hemoglobin and hematocrit was observed in the males and females rats receiving high dose of rHu-EPO. rHu-EPO administration for 30 days showed a marked decrease in glucose concentration. At the highest dose groups, there was a significant incerase in the weights of spleens in both sexes. but this was considered to be related to its pharmacological activity. These results indicate that rHu-EPO does not induce any significant toxic effect on Sprague-Dawley rats for 30 days.

• Reproductive and Developmental Biology
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• 제30권1호
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• pp.27-34
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• 2006

This study was conducted to compare the reproduction ability of the wild type boar and recombinant human erythropoietin (hEPO) transgenic boar semen. Ejaculated boar semen was analyzed by flow cytometry, Elisa and IVF methods. In experiment 1, flow cytometric analysis showed that the live sperm ratio of transgenic boar sperm significantly lower (P<0.05) than that of wild type boar after incubation at 20, 22, 24 and 26 hr. In experiment 2, the presence and levels of various cytokines (IL-6, IL-10 and $TNF-{\alpha}$) to related animal reproduction in the seminal and blood plasma were examined using specific enzyme immunoassay. There was no significant difference between both groups. In experiment 3, the fertilizing capacity and developmental ability of both boar sperm were compared. The transgenic boar sperm had a significantly low capacity of penetration, sperm-zona binding, embryo development, and blastocyst formation compared to wild type sperm (P<0.05). These results suggest that transgenic boar sperm harboring hEPO gene has low sperm viability than wild type boar, and it is a reason to decrease of fertility and litter size.