• 한국수정란이식학회지
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• 제31권3호
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• pp.179-183
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• 2016

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell-derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

• 한국수정란이식학회지
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• 제23권2호
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• pp.67-76
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• 2008

Since the birth of Dolly using fully differentiated somatic cells as a nuclear donor, viable clones were generated successfully in many mammalian species. These achievements in animal cloning demonstrate developmental potential of terminally differentiated somatic cells. At the same time, the somatic cell nuclear transfer (SCNT) technique provides the opportunities to study basic and applied biosciences. However, the efficiency generating viable offsprings by SCNT remains extremely low. There are several explanations why cloned embryos cannot fully develop into viable animals and what factors affect developmental potency of reconstructed embryos by the SCNT technique. The most critical and persuasive explanation for inefficiency in SCNT cloning is incomplete genomic reprogramming, such as DNA methylation and histone modification. Numerous studies on genomic reprogramming demonstrated that incorrect DNA methylation and aberrant epigenetic reprogramming are considerably correlated with abnormal development of SCNT cloned embryos even though its mechanism is not fully understood. The SCNT technique is useful in cloning farm animals because pluripotent stem cells are not established in farm animal species. Therapeutic cloning combined with genetic manipulation will help to control various human diseases. Also, the SCNT technique provides a chance to overcome excessive demand for the organs by production of transgenic animals as xenotransplantation resources. Here, we describe the factors affecting the efficiency of generating cloned farm animals by the SCNT technique and discuss future directions of animal cloning by SCNT to improve the cloning efficiency.

• 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.

• 대한수의학회지
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• 제30권4호
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• pp.355-360
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• 1990

포유동물의 초기 발생단계에서 핵의 분화와 전능성(totipotency) 을 규명하고, 수정란의 cloning technique를 개발하여 우량유전자로 조성된 개체를 복제함으로써 효과적인 종축개량 기법으로 응용하기 위하여 생쥐 수정란을 모델로 하여 미세조작기법과 Sendai virus를 이용한 핵융합기술을 이용하여 인위적으로 동일한 유전자를 가진 복제 수정란을 작출하고 이들의 작출효과, 체외발달능력 및 체내 이식후 개체발생여부 등을 조사하였다. 2-세포기, 4-세포기 및 8-세포기의 수정란으로부터 핵을 채취하여 이들을 탈핵된 2-세포기의 수정란에 이식하였을 때, 이들의 핵융합 성공율은 각각 88.6%, 87.1% 및 84.7%이었다. 나아가서 이들 핵융합된 수정란을 체외에서 96시간 배양한 결과, 2-세포기, 4-세포기 및 8-세포기의 핵이 이식된 수정란은 각각 76.5%, 68.4% 및 48.3%가 배반포로 발달하였다. 핵이식 후 체외에서 배반포로 발달된 수정란을 골라 수란생쥐에 이식하였던 바, 2-세포기의 핵이 이식된 수정란 156개 중 58개(37.1%) 가 발달하여 신생자로 생산되었으며, 4-세포기의 핵아 이식된 수정란 135개 중 40개(29.6%)가, 그리고 8-세포기의 핵이 이식된 92개의 수정란 중 15개(16.3%)가 신생자로 생산되었다.

• 한국원자력학회:학술대회논문집
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• 한국원자력학회 1995년도 추계학술발표회논문집(1)
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• pp.480-486
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• 1995

Turbulent flow field in a subchannel of bare rod bundles has been numerically simulated using the control volume based finite element method. Launder & Ying model of Reynolds stress and Lam & Bremhorst low-Reynolds number model are implemented in k-$\varepsilon$ equations and momentum equations. Secondary flows are simulated using the stream function and vorticity approach. The control volume based finite element method enable to use the upwind scheme (donor cell scheme). Sensitivity of the constants in the models are studied, and proper values are found to get the close result to the measured flow distributions.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.62-62
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• 2001

Recently, production of transgenic animal by nuclear transfer has been known as a useful method. The production of cloned offspring derived from nuclear transfer depends upon a variety of factors such as species, donor cells type and cell cycle, and source of recipient ova. Therefore, we attempted a different transgenic methods using follicular granulosa cells (GCs). In general, ovulated GCs undergoes lutenization and transformation in vitro which might defective effects on developmental potential. In order to avoid the GCs transformation in vitro culture system, we introduced a direct injection of retrovirus into the follicles and then collected them mechanically from ovaries of 6-8 week-old ICR mice. Retrovirus vector constructed with pLN $\beta$ EGFP was injected into the follicles. The follicles are cultured in $\alpha$ -MEM supplemented with human FSH, LH and ITS in Costar Transwell dish for 4 days. Survival rate of virus injected follicles was 52.1% (12/23) and expression rate of EGPP gene was 33.3% (4/12). In this study, we found GCs performed transgenesis in our culture system. In addition, the GCs in follicle may be developed in vivo like environment rather than in vitro environment. Thus, the use of GCs as donor cells may be useful in the nuclear transfer for cloning of genetic modification. Therefore, these results suggest that follicular GCs can be transfected by viral vector during folliculogenesis in vitro.

• Nuclear Engineering and Technology
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• 제41권10호
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• pp.1347-1360
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• 2009

A multi-dimensional component for the thermal-hydraulic system analysis code, MARS, was developed for a more realistic three-dimensional analysis of nuclear systems. A three-dimensional and two-fluid model for a two-phase flow in Cartesian and cylindrical coordinates was employed. The governing equations and physical constitutive relationships were extended from those of a one-dimensional version. The numerical solution method adopted a semi-implicit and finite-difference method based on a staggered-grid mesh and a donor-cell scheme. The relevant length scale was very coarse compared to commercial computational fluid dynamics tools. Thus a simple Prandtl's mixing length turbulence model was applied to interpret the turbulent induced momentum and energy diffusivity. Non drag interfacial forces were not considered as in the general nuclear system codes. Several conceptual cases with analytic solutions were chosen and analyzed to assess the fundamental terms. RPI air-water and UPTF 7 tests were simulated and compared to the experimental data. The simulation results for the RPI air-water two-phase flow experiment showed good agreement with the measured void fraction. The simulation results for the UPTF downcomer test 7 were compared to the experiment data and the results from other multi-dimensional system codes for the ECC delivery flow.

• 한국가축번식학회지
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• 제20권4호
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• pp.459-465
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• 1997

본 연구는 핵이식의 시기가 융합과 체외발달에 미치는 영향을 조사하고 체외수정 공핵배의 핵과 체외성숙난자간에 전기융합의 최적 통전전압과 통전시간을 결정하기 위하여 시도하였다. 수핵난자는 체외성숙후 25~27시간에 제핵하였고 난자의 추가성숙을 위하여 융합전에 18~20시간 더 배양하였다. 공핵체외수정란은 16~32세포기까지 BOEC와 공배양하였다. 공핵배의 할구 이식시간은 조기핵이식구에서는 체핵후 1~3시간, 자연핵이식구를 체핵후 16~18시간이었다. 융합은 체외성숙후 43~45시간에 행하였다. 융합율은 할구 이식시기에 따라 차이가 없었으나 난할율과 8~16세포기 발달은 자연핵이식구에서 높았다. 융합, 난할 및 상실배 배반포 발달율은 1.0kV/cm보다 0.75kV/cm전압에서 높았으며 0.7kV/cm에서 상실배 배반포율은 17.6%였다. 통전시간에 따라 융합율은 차이가 없었으나 50$\mu$sec와 70$\mu$sec에서 난할율과 상실배-배반포 발달율이 다소 높았다. 본 결과에서 핵이식의 최적시기는 수핵난자의 체외성숙후 42~44시간이었으며 최적 전기융합 조건은 50~70$\mu$sec의 1회 0.75kV/cm임을 알 수 있었다.

• 한국수정란이식학회지
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• 제13권1호
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

• Toxicological Research
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• 제16권1호
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• pp.1-8
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• 2000

Astrocytes generate free radicals including nitric oxide (NO) and reactive oxygen intermediates(ROI) which in turn play roles in the pathogenesis of degenerative diseases and sclerotic changes of the brain. This study was designed to evaluate the mechanism that free radicals contribute to the cytotoxicty of rat neonatal primary astrocytes. Treatment with NO donors alone including soldium nitroprusside(SNP), S-nitrosoglucathinoe (GSNO), and S-nitroso-n-acetylpenicillamine (SNAP) showed a little effect on the death of rat neonatal primary astrocytes, whereas SNP markedly induced the death of RAW 264.7 cells. ROI inculding H2O2 and O2 donor also slightly induced the death of rat primary astrocytes. However, 3-morpholinosydnonimine(SIN-1), a donor of peroxynitrite (ONOO), which is a reactive compound of NO with superoxide, significantly decreased the viability of rat primary astrocytes in a dose-dependent manner. Cells were retarded in outgrowth of viability of cellular processes with cell shrinkage and detachment from culture dishes. Hoechst staining demonstrated that SIN-1-induced cell death might be due to an apoptosis which was characterized by nuclear condensation and fragmentation. SIN-1-induced apoptosis was prevented by the pretreatment with superoxide dismutase (SOD) and catalase in rat primary astorocytes. Furthermore, prevention of the generation of reduced glutathione (GSH) by DL-buthionine-[S, R]-sulfoximine (BSO) aggravated the cytotoxic effects of SNP, benzene triol, and SIN-1 in rat primary astrocytes. Taken together, it is suggested that peroxynitrite may be a major effector of apoptosis and cellular antioxidant system is important for cell survival in rat prima교 astrocytes.