• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.113-113
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• 2003

This study was conducted to investigate the effect of vitrification solution(VS) on in vitro developmental competence of immature porcine oocytes. The immature porcine oocytes were exposed to the following vitrification solution, at RT. 1) EFS sol. : 20% ethylene glycol (EG) 3 min, 40% EG + 18%(w/v) Ficoll(MV70, 000) + 0.3 M sucrose 30 sec, 2) GE sol. : 10% glycerol 5 min, 10% G + 20% EG 5 min, 25% G +25% EG 30 sec, 3) EG sol : 1.5M EG 2.5 min, 5.5 M EG + 1.0 M sucrose 30 sec. Oocytes were immediately transferred into 1.0 M, 0.5 M, 0.25 M, 0125 M, 0 M sucrose solution for 2.5 min each at RT. After removal of VS, immature oocytes were matured in vitro and subsequently all oocytes were subjected to IVF followed in vitro culture for 7 days. Maturation rates of oocytes were 38.8%, 44.5%, 22.4% and 57.6%, in EFS, EG, GE and Control, respectively, maturation rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.01). Fertilization rates of oocytes in Control was significantly higher than other treated groups(P<0.05), but no difference were observed among treated groups. Polyspermic rates were no significant difference among four groups. Cleavage rates of oocytes were 21.9%, 47.1%, 19.0% and 65.9%, in EFS, EG, GE and Control, respectively, cleavage rates of oocytes in EG and Control was significantly higher than EFS and GE(P<0.05), but blastocyst formation rates were no significant difference among four groups. These results suggested that the use of EG solution could be a great challenge for reaching a successful vitrification of immature porcine oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제38권1호
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• pp.24-30
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• 2011

Objective: The objectives of this study were to analyze efficacy of immature and mature mouse oocytes after vitrification and warming by applying various combinations of cryoprotectants (CPAs) and/or super-rapid cooling using slush nitrogen ($SN_2$). Methods: Four-week old ICR female mice were superovulated for GV- and MII-stage oocytes. Experimental groups were divided into two groups. Ethylene glycol (EG) only group: pre-equilibrated with 1.5 M EG for 2.5 minutes and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 seconds. EG+dimethylsulfoxide (DMSO) group: pre-equilibrated with 1.3 M EG+1.1 M DMSO for 2.5 minutes and equilibrated with 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 20 seconds. The oocytes were loaded onto grids and plunged into $SN_2$or liquid nitrogen ($LN_2$). Stored oocytes were warmed by a five-step method, and then their survival, maturation, cleavage, and developmental rates were observed. Results: The EG only and EG+DMSO groups showed no significant difference in survival of immature oocytes vitrified after warming. However, maturation and cleavage rates after conventional insemination were greater in the EG only group than in the EG+DMSO group. In mature oocytes, survival, cleavage, and blastocyst formation rates after warming showed no significant difference when EG only or EG+DMSO was applied. Furthermore, cleavage and blastocyst formation rates of MII oocytes vitrified using $SN_2$ were increased in both the EG only and EG+DMSO groups. Conclusion: A combination of CPAs in oocyte cryopreservation could be formulated according to the oocyte stage. In addition, $SN_2$ may improve the efficiency of vitrification by reducing cryoinjury.

• 한국발생생물학회지:발생과생식
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• 제15권4호
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• pp.281-289
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• 2011

돼지 중간엽 줄기세포를 Dimethyl sulfoxide(DMSO), Ethylene glycol(EG), 그리고 DMSO/EG을 이용하여 세포동결을 유도한 후 적절한 동결보호제를 알아보았다. 2개월 이내 돼지 골수에서 중간엽 줄기세포를 분리하여 colony 형성 및 alkaline phosphatase(AP) 활성을 확인하고, 지방 세포로의 분화 유도에 의한 줄기세포의 능력을 확인하였다. 이들 중간엽 줄기세포의 완만 동결을 위해, DMEM에 각각 10% DMSO, 1.5M EG, 5% DMSO/0.75M EG의 동결보호제를 섞은 후 cryovial에 넣고, cryo-containe를 이용하여 $25^{\circ}C$에서 $-80^{\circ}C$까지 $-1^{\circ}C$/min 속도로 동결하였다. 일주일간 저장 후 세포의 생존률은 미동결 세포는 동결 세포군보다 유의적으로 높음을 확인할 수 있었으나, 동결 처리군 간에는 차이가 없었다. 줄기세포 유지 유전자인 Sox-2와 Nanog 발현은 동결 후 배양 시간에 따라 발현량이 증가하는 경향을 보였으나, 동결처리군 간에는 유의적인 차이가 없었다. 세포사 관련 유전자인 Bax의 발현은 모든 군에서 비슷하였다. 또한 지방, 연골 및 뼈세포 분화와 관련된 유전자의 발현은 동결 전 세포와 동결 후 세포군에서 비슷한 경향을 보였다. 이러한 결과는 돼지중간엽 줄기세포 동결함에 있어서 10% DMSO, 1.5MEG, 5% DMSO/0.75M EG 모두 적절한 동결보호제로 이용할 수 있음을 시사한다.

• Journal of Animal Science and Technology
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• 제48권6호
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• pp.797-804
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• 2006

본 연구에서는 동결 보호제 EG l에 sucrose 첨가 농도에 따른 생존성의 실험의 결과를 요약하면 다음과 같다. 1.5 M EG와 1.8 M EG 만을 이용하여 동결융해 후 생존서의 조사한 결과 71.1%와 70.2%로 각각 나타났다. 총세포수에 있어서도 127±1.3개와 124±1.6개로 생존율과 총세포수에 있어서도 두 그룹간에는 유의적인 차이를 보이지 않았다. 1.5 M EG와 1.8 M EG에 0.1 M sucrose를 각각 첨가한 후 동결 보존하여 융해 하였을 때 생존율과 총세포수 조사 결과는 1.5 M EG에 0.1 M sucrose 처리구가 73.6% 그리고 1.8 M EG 에 0.1 M sucrose 첨가군은 76.9%의 결과를 보였으며 총세포수 에 있어서도 118±1.2 와 112±1.2 개의 결과를 보여 생존성과 총세포수에 있어서도 두 처리군 모두 유의적인 차이를 나타내지 않았으나 1.5 M EG 처리구에서 총세포수는 다소 높은 경향을 보였다. 1.5 M EG 와 1.8 M EG에 0.3 M sucrose를 첨가하여 각각 생존성과 총세포수 조사 결과는 70.8%와 88.7%의 생존율을 나타내어 1.8 M EG 에 0.3 M sucrose 처리구가 유의적으로(P<0.05) 높은 결과를 보였다. 따라서 소 체외수정란을 conventional slow-freezing 방법으로 동결 보존할 경우는 1.8 M EG 동결보호제에 0.3 M sucrose를 병행하여 사용하는 방법이 수정란을 최상의 상태로 유지할 수가 있어 수정란이식에 적용할 경우 효과적일 것으로 사료된다.

• 한국수정란이식학회지
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• 제27권3호
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• pp.155-162
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• 2012

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY ($69.00%{\pm}4.18$; EG and $63.00%{\pm}9.75$; 7% G) than 8% LDL ($57.00%{\pm}5.70$; EG and $52.00%{\pm}4.47$;G). Treatment of 4% LDL + 5% EY-EG ($66.85%{\pm}5.06$) has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG ($64.65%{\pm}6.10$) among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

• KSBB Journal
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• 제13권5호
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• pp.518-523
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• 1998

A large quantity of ethylene glycol(EG) is remained in the effluent after pretreating polyester weight-loss wastewater physicochemically in the fist stage and must be treated biologically in the second stage. Therefore, an excellent EG-utilizing bacteria strain was isolated from the natural system and the optimal culture conditions of the strain were investigated. The optimal culture conditions of temperature, pH, and nitrogen source were found to be 35$^{\circ}C$, 7.5 and ammonium chloride, respectively, when CODCr removal efficiency was more than 90%. The growth of stains and EG removal efficiency was slightly improved by adding elements such as niacin and biotin. With increasing inoculation size in a batch culture, the removal efficiency of EG was conspicuously increased. Growth rate was inhibited when the initial concentration of EG was more then 30g/L. The strain was identified as Pseudomonas sp. based on morphological and biological characteristics and named as Pseudomonas sp. EG1.

• 한국수정란이식학회지
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• 제27권4호
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• pp.253-258
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• 2012

This study was carried out to effects of ethylene glycol concentration, sucrose and culture day of in vitro production embryo on slow-down freezing in Hanwoo. 6, 7, 8 and 9 day embryos produced in vitro were frozen using 1.8M EG+0.1M sucrose, 1.8M EG+0.5% BSA and 1.5M EG+0.1M sucrose media. Survivability was confirmed after frozen-thawed 24 and 48h and ICM, TE cell number were counted by Hoechst 33342 and PI staining after frozen-thawed 24h. As a result, 1.8M EG+0.1M sucrose group was most significantly (p<0.05) higher compared with the other treatment groups on survivability, TE and total cell number after frozen-thawed 24h ($94.2{\pm}2.6%$, $94.67{\pm}3.4$ and $129.67{\pm}5.5$). ICM number did not found significant (p<0.05) differences between the three treatment groups. in 6, 7, 8 and 9 day of embryos using three types of freezing media, frozen-thawed, 1.8M EG+0.1M sucrose groups with embryos cultured 8 day was significantly (p<0.05) highest survivability to $98.3{\pm}1.7%$ after frozen-thawed 24h. 1.5M EG+0.1 sucrose group with embryos cultured 9 day was significantly higher survivability than group of embryos cultured 8 day after frozen-thawed 24 and 48h. In conclusion, 1.8M EG+0.1M sucrose media is considered to be effective to cryopreservation of embryos cultured 8 and 9 day.

• 식물병연구
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• 제19권2호
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• pp.108-113
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• 2013

Ralstonia solanacearum에 의한 시들음 피해는 고온기 재배가 증가함에 따라 피해가 증가하고 있다. 이에 재배적 방법으로 토마토대목을 이용하고 있으나 병이 다 발생하는 지역에서는 효과가 기대에 미치지 못한다. 이에 따라서 AVRDC에서 수집한 풋마름병 저항성 가지 대목인 EG203을 재료로 하여 풋마름병 다 발생포장인 부여 토마토시험장 비닐하우스에서 2003-2005년, 구미시 비닐하우스에서 2009-2011년까지 두 곳에서 각각 3년간 실시하였다. EG203(가지)는 광발아 종자로 파종 시 육묘 트레이에 직접 파종하여야 하며 EG-203(가지)의 파종 적기는 토마토 접수보다 3주전에 파종해야 접수와 비슷한 경경(2.5-3.0 mm)에 도달하여 접목하기에 알맞았다. 접목방법은 맞접과 삽접이 93-96%의 활착률을 보여 대목으로 사용가능한 것으로 확인되었다. 접목 후 포장에 정식한 후의 풋마름병 발생을 2003년부터 2005년까지 3년 동안의 평균은 EG203(가지) 접목 시 4.3%로 실생의 58.0%보다 매우 낮았으며 대목용 품종의 25.0-36.7%보다도 낮게 나타났다. 구미에서 2009년부터 2011년까지 3년간 대목 효과에 대하여 시험한 결과는 2009년 5농가에서 EG203과 실생묘를 비교한 결과 EG203에서는 풋마름병 발생률이 2-5%였으나 실생은 20-80%를 보였다. 2010년에는 토마토대목('B-blocking', '청강')과 가지대목(EG203)을 갖고 비교 한 결과 풋마름병 발생률의 차이는 보이지 않았으나 가지대목(EG203)에서 복화방 출현이 늦어 수확이 감소되는 경향을 보였다. 2011년 토마토대목('청강')과 가지대목(EG203)으로 3농가에서 시험한 결과 토마토대목('청강')에서 풋마름병 발생률은 60-85%, 가지대목(EG203)은 0-1%로 가지대목에서 병발생률이 매우 낮았다. 가지대목(EG203)을 이용할 경우 토마토대목과 비교 복화방이 늦게 발생하여 토마토 생산량이 줄었다. 따라서 병 발생이 낮은 곳에서는 토마토대목을 활용하여 접목을 하고 병이 다 발생하는 지역에서는 가지대목(EG203)을 활용하는 것이 재배에 유리하다고 판단되어진다.

• Journal of Microbiology and Biotechnology
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• 제22권3호
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• pp.390-399
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• 2012

Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa and isoelectronic point (pI) of 5.29. The expression of gene egIV in T. viride AS 3.3711 could be induced by sucrose, corn straw, carboxymethylcellulose (CMC), or microcrystalline cellulose, but especially by CMC. The transcripts of egIV were regulated under these substrates, but the expression level of the egIV gene could be inhibited by glucose and fructose. Three recombinant vectors, pYES2-xegIV, $pYES2M{\alpha}$-egIV, and $pYES2M{\alpha}$-xegIV, were constructed to express the egIV gene in Saccharomyces cerevisiae H158. The CMCase activity of yeast transformants $IpYES2M{\alpha}$-xegIV was higher than that of transformant IpYES2-xegIV or $IpYES2M{\alpha}$-egIV, with the highest activity of 0.13 U/ml at induction for 48 h, illustrating that the modified egIV gene could enhance CMCase activity and that $MF{\alpha}$ signal peptide from S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae. The recombinant EGIV enzyme was stable at pH 3.5 to 7.5 and temperature of $35^{\circ}C$ to $65^{\circ}C$. The optimal reaction condition for EGIV enzyme activity was at the temperature of $55^{\circ}C$, pH of 5.0, 0.75 mM $Ba^{2+}$, and using CMC as substrate. Under these conditions, the highest activity of EGIV enzyme in transformant $IpYES2M{\alpha}$-xegIV was 0.18 U/ml. These properties would provide technical parameters for utilizing cellulose in industrial bioethanol production.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2001년도 제18차 정기총회 및 학술발표 PROCEEDINGS
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• pp.40-46
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• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.