• Reproductive and Developmental Biology
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• 제31권2호
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• pp.127-131
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• 2007

Embryonic germ (EG) cells are undifferentiated stern cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of differentiation both in vitro and in vivo have been established. Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide inexhaustible source of karyoplasts in nuclear transfer (NT). In this study the efficiencies of NT using porcine EG and fetal fibroblast cells were compared. Two different techniques were used to perform NT. With conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes, the rates of development to the blastocyst stage in EG and somatic cell NT were 16.8% (59/351) and 14.5% (98/677), respectively. In piezo-driven microinjection (Honolulu method) of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 9.4% (9/96), respectively. Regardless of NT methods used in this study, EG cell NT gave rise to comparable rate of blastocyst development to somatic cell NT. Overall, EG cells can be used as karyoplast donor in NT procedure, and embryos can be produced by EG cell NT that may be used as an alternative to conventional somatic cell NT.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.103-108
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• 2007

This study was carried out to investigate the effects of cryoprotectants, warming solution and removal of lipid on open pulled straw vitrification (OPS) method of porcine embryos produced by nuclear transfer (NT) of fetal fibroblasts. All solutions used during vitrification were prepared with holding medium consisting of 25 mM Hepes buffered TCM199 medium containing 20% fetal bovine serum (FBS) at $38.5^{\circ}C$. The blastocysts derived from NT with or without lipid were vitrified in each medium of different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Also, blastocysts after cryopreservation were warmed into different concentrations of sucrose in warming solution. The optimal concentrations of cryoprotectants in vitrification solution were 10% DMSO + 10% EG in vitrification solution 1 (VS1) and 20% DMSO + 20% EG in vitrification solution 2 (VS2). The optimal concentrations of sucrose were 0.3 M sucrose in warming solution 1 (WS1) and 0.15 M sucrose in warming solution 2 (WS2). lipid removal from oocytes before NT enhanced the viability of NT embryos after vitrification. Our results show that use of the OPS method in conjunction with lipid removal provides effective cryopreservation of porcine nuclear transfer embryos.

• 한국수정란이식학회지
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• 제27권2호
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• pp.113-119
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• 2012

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1178-1188
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• 2014

In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.78-78
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• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• 한국가축번식학회지
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• 제26권3호
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• pp.245-252
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• 2002

본 연구는 체세포 핵치환에 탈핵 후 통격한 소미수정란을 사용함에 있어서, MVC 초자화 동결방법과 탈핵난자의 활성화시기가 융해 후 생존율과 핵치환 이후 체외 발달에 미치는 영향을 조사하고자 실시하였다. 체외에서 20시간 동안 체외성숙된 소 미수정란은 수핵란으로 사용하기 위하여 5$\mu\textrm{g}$/$m\ell$ hoechst 처리 후, 형광현미경하에서 핵을 제거하였다. 본 실험은 세 그룹으로 나누어 실행되었다 Group I은 동결하지 않고 핵치환을 한 대조군이며, group III와 group II는 핵이 제거된 난자를 MVC 방법으로 동결하기 전과 후에 활성화 처리 (5$\mu\textrm{m}$의 ionomycin에 의해서 5분간 처리) 한 군이다. 초자화 동결을 위해서는 group II와 group III의 탈핵란은 EG10에서 5~10분간 전처리하고 EG30에서 30초간 노출하여 액체 질소에 침지하였다. 융해는 37$^{\circ}C$에서 4단계로 이루어졌다. 실험군은 모두 소 귀세포를 이용하여 핵치환을 실시하였으며, 전핵을 유도하기 위한 활성화를 위해서는 10$\mu\textrm{g}$/$m\ell$ cycloheximide와 2.5$\mu\textrm{g}$/$m\ell$ cytochalasin D)가 첨가된 CRlaa 배양액에서 1시간, 이후 10 $\mu\textrm{g}$/$m\ell$ cycloheximide가 들어있는 CRlaa 배양액에서 4시간동안 배양하였다. 활성화 처리가 끝난 난자들은 CRlaa 배양액에서 2일간 배양하여 난할이 유도된 난자만을 선별하여 난구세포와 7일 동안 공배양하였다. 동결 융해 이후 group II와 group III의 탈핵된 소 미수정란의 체외 생존율은 81.0%와 84.9%로 유의적인 차이가 없었다. 체세포와 수핵란과의 융합율도 각각 69.0%와 70.0%로 대조군 (75.2%) 과도 유의적인 차이를 나타내지 않았다. 난할율은 53.4%와 58.4%로 group II와 group III간에 유의적인 차이를 나타내지 않았지만 group II의 분할된 세포질을 가진 이상난자의 비율이 group III보다 유의하게 높게 나타났다 (P<0.05). 또한, morula 이상으로 발달율도 group II (8.6%) 에서 group III (15.6%)보다 낮은 결과를 얻었다 하지 만 group III (15.6%)의 체외 발달율은 대조군 (24.8%)과 유의한 차이를 없었다. 따라서, MVC 동결 방법은 탈핵된 소 미수정란을 동결하기에 적합한 방법이며, 탈핵 후 activation을 유도하고 초자화 동결한 난자는 동결하지 않은 신선란과 동일하게 체세포 핵치환에 유용하게 이용될 수 있으리라 사료된다.