• 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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.6
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• pp.745-752
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• 2007

This study examined the effects of different levels of evening primrose oil (EPO) on the accumulation of ${\gamma}$-fatty acids in broiler meat. Six hundred one-day-old male chicks (Ross strain) from commercial broilers were divided randomly into 6 groups${\times}$4 repeat pens. The broilers were fed experimental diets containing 4.0% tallow (control), 0.5% EPO, 0.7% mixed oil (EPO 70:soy bean oil 30), 1.5% EPO, 3.0% EPO or 4.0% EPO for two weeks of broiler finisher. There was a significant difference in body weight gain between the control and treatment groups except for the 0.5% EPO group (p<0.05). There was a significant difference in the percentage of thigh and breast weight against the carcass weight between control and treatment groups except for the 0.5% EPO group in the thigh and 0.5% EPO and 4.0% EPO groups in the breast weight (p<0.05). The saturated fatty acid levels of the skin and breast muscle lipid of the broilers fed diets containing EPO were significantly lower than that of the control group (p<0.05), while the level of unsaturated fatty acid was significantly higher than that of the control group (p<0.05). The ${\gamma}$-fatty acid (GLA, gamma.linolenic acid, 18:3n-6) level was particularly higher in the chicken meat lipids from the broilers fed EPO than in the control group (p<0.05). This shows that feeding EPO to chicks can produce novel functional broiler meat that is enriched in gamma-linolenic acid.

• Biomedical Science Letters
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• v.15 no.2
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• pp.161-166
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• 2009

Human erythropoietin(EPO) is a glycoprotein that enhances red blood cell production by stimulating proliferation and differentiation of erythroid progenitor cells in the bone marrow. Patients with chronic kidney disease(CKD) suffer from anemia caused by reduced production of EPO in the kidney. Recombinant human EPO protein has been used successfully for the treatment of anemia associated with CKD. Recently, attention has been paid to the development of side effect of EPO, pure red cell aplasia(PRCA), in some patients with CKD. PRCA is a rare disorder of erythropoiesis that leads to a severe anemia due to an almost complete cessation of red blood cell production. EPO-related PRCA is caused by the production of EPO-neutralizing antibodies(Abs) that eliminate the biological activity of EPO as well as endogenous EPO in patients undergoing therapy. Since 1988, almost 200 cases worldwide have been reported with Ab-positive PRCA after receiving EPO therapeutics. The underlying mechanisms of the breaking of immune tolerance to self-EPO have been investigated. Modification of formulation, organic compounds of container closures, and route of administration has been suggested for the possible mechanism of increased immunogenicity of EPO. A number of assays have been used to detect Abs specific to EPO. These assays are generally grouped into two major categories: binding Ab assays and neutralizing Ab assays(bioassays). There are several types of binding Ab assays, including radioimmunoprecipitation assay, enzyme-linked immunosorbent assay, and the BIAcore biosensor assay. In vitro cell-based bioassays have been utilized for the detection of neutralizing Abs. Finally, the recent experience with anti-EPO Abs may have considerable implications for the future development and approval of EPO preparations. Also, considering that millions of patients are being treated with EPO, clinicians need to be aware of signs and consequences of this rare but severe clinical case.

• Biomolecules & Therapeutics
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• v.2 no.4
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• pp.343-346
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• 1994

In vivo activity of recombinant human erythropoietin (rh-EPO) has been examined using polycythemic model in mice and acute hemorrhage model in rats. The number of reticulocytes in blood stream was increased after a single injection of rh-EPO depending on the dosage of rh-EPO in polycythemy model. It seemed that optimal dose of rh-EPO for polycythemic mice was around 1-10 U/kg. Rh-EPO also showed the effectiveness for increase of reticulocyte numbers both in male and female rats after bleeding. The number of reticulocytes and the change of hemoglobin concentration in the blood stream of normal rats has been examined after injection of rh-EPO. The maximum value of reticulocyte was observed on the 6th day of the injection in these normal rats. In addition, the increase of reticulocyte and the concentration of hemoglobin were dependent on the dosage of rh-EPO. The increase of hemoglobin concentration was continued to the 9th day after injection. In this study, the efficacy of rh-EPO was confirmed in both mice and rats.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.55-55
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• 2001

Erythropoietin(EPO)는 적혈구 세포 증식, 분화 및 생존에 있어서 가장 중요한 요인이다. 또한, 빈혈성저산소증에 있어서도 EPO가 중요한 역할을 한다고 알려져 있다. 태아에서 EPO 생산부위는 간이라고 알려져 있으나, 임신 120-140일에 신장으로 이동하기 시작하여 출생 후 약 40일경 이후에는 완전히 신장에서만 분비한다 EPO단백질의 분비는 오전 8시에 가장 낮고 오후 8시에 가장 높은 2중 리듬의 형태로 발현되어진다. EPO는 27개의 leader sequence와 165개의 아미노산으로 총 193개의 아미노산으로부터 분비된다. EPO단백질의 분자량은 18 kDa이나, 약 40%의 당쇄가 첨가되어있는 당단백질으로서 분자량은 30 kDa이다 N-linked 당쇄 3개(Asn-24, 38 및 83)와 O-linked 당쇄 1개(Ser 126)의 첨가부위가 존재하며, 2개의 disulfide bridges(7-161번, 29-33번)를 형성하고 있다. 이러한 당쇄의 수식은 EPO의 대사에 있어서 매우 중요하다. EPO를 가축의 소변으로부터 생산하기 위하여 생쥐의 3.6 kb UII promoter 하류에 genome hEPO와 SV 40 poly A를 연결하여 형질전환용 발현 벡터를 구축하였으며, 과배란 유기로 채란되어진 돼지의 1-세포기 수정란의 웅성전핵에 유전자를 미세주입기로 주입 후 즉시 대리모에 이식하였다. 66두에 미세주입된 1572개의 수정란을 외과적 방법으로 이식, 평균 23개의 수정란을 이식하였다. 생산된 자돈 112두중 2두(3-5, 3-15번)에서 PCR양성반응(304, 567bp)을 나타내어, 2두의 돼지로부터 소변을 회수하였다. 회수된 소변을 이용 Elisa방법으로 EPO를 분석한 결과 3-5번 돼지에서만 분만 후 지속적으로 EPO농도가 증가되었다. EPO의 최고농도는 1.1 IU/$m\ell$였으며, 이러한 결과는 CHO 세포에서의 500-1000 IU/$m\ell$의 생산량보다도 약 500-1000배정도 낮은 수준이었다. 이상을 종합하여 보면, 1) 가축에서도 생리활성물질을 소변에서 생산할 수 있는 UII promoter의 활용가능성을 제시하였으며, 2) 현재로서는 EPO의 발현량이 너무 낮아, 사용된 생쥐의 promoter를 보완할 필요성이 있다고 사료된다. 그러나, UII promoter를 이용하여 생리활성 물질을 생산할 수 있는 형질전환 돼지 생산의 성공은, 앞으로 형질전환 가축을 이용하는 활용 면에서도 더욱 더 활발할 것으로 기대된다.

• Toxicological Research
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• v.13 no.1_2
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• pp.149-152
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• 1997

This test was performed to evaluate the ocular and skin irritation of EPO (Erythropoietin). The results as follows: 1. Ocular irritation test There were no observed clinical signs, body weght changes by EPO during experimental period. The acute ocular irritation index(A.O.I.), mean ocular irritation index(M.O.I.) and Day-7 individual ocular irritation index(I.O.I.) of EPO at dose of 1000U and 10, 000U were 0, respectively. Therefore we evaluated that EPO was non-toxic to eyes. 2. Skin irritation test There were no observed clinical signs, body weght changes and gross pathologic findings by EPO during experimental period. There were no observed erythema, eschar formation and edema formation on intact and abraded skin treated by EPO. The primary irritation index(P.I.I.) of EPO at dose of 1000U and 10, 000U were 0, respectively and were evaluated none irritating product about skin irritation.

• Environmental Mutagens and Carcinogens
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• v.16 no.2
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• pp.103-108
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• 1996

The mutagenic potential of rHu-EPO was evaluated using the short-term genotoxicity tests including Ames, chromosome aberration and micronuclei tests. In Salmonella typhimurium assay, rHu-EPO did not show any mutagenic response in the absence or presence of S9 mix with TA98, TA100, TA1535, and TA1537. In chromosome aberration test, rHu-EPO did not show any significant effect on Chinese Hamster Ovary(CHO) cells compared with control. In micronucleus test using male ICR mice, a dose-dependence increase in the frequency of micronucleuted polychromatic erythrocytes(MNPCEs) was observed in bone marrow cells treated with rHu-EPO. However, it was related to the secondary effect of rHu-EPO and the number of MNPCEs was equal to spontaneous frequency. These results indicate that rHu-EPO does not show any positive response in short-term genotoxicity assays.

• 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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• 2001.03a
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• pp.57-57
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• 2001

Erythropoietin(EPO)는 혈액의 구성성분 중에서 적혈구 세포 증식에 중요한 기능을 한다고 알려져 있으며, 최근에는 암, 에이즈의 치료 등에도 효과가 있는 것으로 확인되고 있다. 따라서 본 연구팀은 지금까지 hEPO 유전자를 이용하여, 형질전환 돼지 "새롬이"의 생산에 성공한바 있다. 새롬이의 정액을 활용하여 인공수정을 실시 새롬이의 Fl를 24두 생산하였다. 이에 대하여 "형질전환 돼지의 계대번식시 유전자 전이효율에 관한 연구"라는 제목으로 발표 할 예정이며, 형질전환에 사용된 promoter가 WAP이므로, Fl이 임신, 분만을 하여야만 유즙을 통하여 hEPO물질을 생산할 수 있다. 따라서, Fl(♂)$\times$Fl(♀)의 교배에 의하여 5두가 임신, 분만을 하였으며, 이들 중 1두는 분만 후 21일에 폐사하였으며, 나머지는 현재 정상적으로 사육되고 있다. 이들 5두에 대하여 분만 후 유즙을 채취하여 유즙속에 EPO의 발현여부를 검토하였다. EPO-ELISA kit(medac)를 사용하여 분석결과, 유즙을 8,000배로 희석을 하여야만 Standard curve(1.25~160 mIU/$m\ell$)안에서 EPO의 단백질 발현을 검출할 수 있었다. 5두의 각각 농도는 28, 58, 17, 37, 27 IU/${\mu}\ell$ 였다. 또한 cDNA EPO와 genome EPO를 CHO 동물세포에서 생산하여 10배로 농축한 결과 5.5와 11 IU/${\mu}\ell$의 농도로 유즙과 비교하면 약 20~30배의 낮은 발현양을 나타내었으며, 또한 이러한 결과는 소변에서의 결과(1.1 IU/$m\ell$)보다는 약 30,000배 이상 높은 발현량을 화인 할 수 있었다. 현재, 이들 유즙 물질을 활용 빈혈 질환실험동물을 이용하여 생리활성을 검정, 체내에서 metabolic clearance rate(MCR)를 검토 중에 있다. 또한 F2의 자돈생산은 모돈 5두에서 총 25두가 생산되었는데, 이중 20두 약 80%가 EPO 유전자의 전환율을 나타내었다. 이상을 종합하면, 1) 돼지이용 생리활성물질(EPO)을 유즙에서 대량으로 생산할 수 있는 system의 활용가능성을 국내에서 처음으로 확인하였으며, 2) EPO에 있어서는 국제적으로도 형질전환 가축생산은 최초로 성공하였으며, 현재로서는 생산되어진 물질의 정제수준에 따라 활용가치가 결정되어 질 것으로 사료된다. 생리활성 물질을 생산할 수 있는 형질전환 돼지 생산의 성공은, 앞으로 형질전환 가축생산 뿐 만 아니라, 장기이식 및 복제돼지 생산의 활용 면에서의 응용가능성이 기대된다.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.3
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• pp.450-458
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• 2006

The present study was carried out to investigate the changes in chemical and sensory properties, and cholesterol lowering effect of evening primrose oil (EPO) addition in cholesterol-reduced Cheddar cheese. The cholesterol removal rate reached 92.07% by ${\beta}$-cyclodextrin in the cheese before EPO addition. The thiobarbituric acid (TBA) value of cholesterol-reduced and EPO-added cheese increased with both ripening time and amount of EPO addition. Addition of 5% EPO resulted in a significant difference in TBA value after 4-week ripening, compared with no addition of EPO. The production of short-chain free fatty acids (FFAs) increased with ripening period in all treatments. From 4 week of ripening, the amounts of short-chain FFA in 3 and 5% EPO-added groups were significantly higher than those in other groups. Among sensory characteristics, rancidity was mostly affected by EPO addition, however, the rancidity value of 1% EPO-added was not significantly different from that of EPO-free and cholesterol-reduced cheese. Also, Cheddar cheese flavor was not profoundly affected by 1% EPO addition in all ripening periods. Total blood cholesterol dramatically decreased from 184.0 to 137.1 mg/dL with 5% EPO-added and cholesterol-reduced cheese following 8 weeks of feeding. The present results indicated that 5% EPO addition resulted in a profound lowering effect on blood total cholesterol with some adverse effects on chemical and sensory properties.