• 한국수정란이식학회지
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• 제31권3호
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• pp.161-168
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• 2016

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.

• 한국수정란이식학회지
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• 제21권2호
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• pp.137-146
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• 2006

본 연구는 공여 세포의 종류, 수핵 난자의 유래 및 수란 산양 발정 동기화 조건이 복제 산양 생산에 미치는 영향을 알아보고자 실시되었다. 공여 세포는 귀 유래 섬유아세포를 분리 배양하여 사용하였으며, 체내 성숙 난자는 성숙한 미경산 재래 산양에 과배란을 유기하여 외과적인 방법으로 난관 관류를 통해 회수하였으며, 배란이 되지 않은 난포란은 난포로부터 흡입 채취한 후, 22시간 동안 체외 성숙을 실시하여 사용하였다. 핵이식은 zona drilling 후, 극체와 세포질 일부분 제거를 통해 제핵을 실시하고, 핵이 제거된 난자의 위란강 내로 공핵 세포를 도입하여 실시하였다. 핵이식란의 융합은 전기 자극 방법으로 실시되었으며, 융합이 완료된 핵이식란의 활성화처리는 핵이식 3시간 후에 Ionomycin과 6-DMAP를 병용 처리하여 실시하였다. 복제 수정란의 체외배양은 0.8% BSA가 첨가된 mSOF 배양액으로 $2{\sim}4$ 세포기까지 체외 배양을 실시한 다음 수란 산양의 난관에 외과적으로 이식하였다. 임신 진단은 초음파 임신 진단기로 이식 후 제 30일과 60일에 실시하였다. 귀세포를 공핵 세포로 사용하였을 때 융합율이(63.8 VS. 26.5%) 태아 세포를 사용했을 때보다 유의적으로 높았다(p<0.05). 총 102개의 복제 수정란을 20두에 이식하였으며, 30일에 4두(20.0%)가 임신하였으며 이중 1두가 1두의 복제 산양을 생산하였다. 수란 산양과 공란 산양간의 발정 동기화 간격(${\pm}0$ 또는 +12시간)별 수태율은 각각 18.2 및 16.7%로서 유의적인 차이가 없었다. 수란 산양의 발정 유기 방법에 따른 수태율에 있어서 CIDR 제거 후 hCG 및 PMSG를 투여하였을 때는 25%가 수태하였으나, hCG만 투여한 수란 산양은 수태가 되지 않아 인위적으로 발정 동기화된 수란 산양을 이용하였을 때는 산자를 생산하지 못했다. 자연 발정 산양의 경우, 발정이 동기화된(${\pm}0$) 수란 산양 1두에 체내 성숙 난자를 수핵란으로 사용하여 생산한 5 개의 복제 수정란을 이식하여 149일만에 국내에서 처음으로 복제 산양(진순이) 생산에 성공하였다. 체외 성숙 난자를 수핵란으로 사용하여 생산된 복제 수정란 5개를 이식하였으나 수태가 이루어지지 않았다. 이상의 결과로 볼 때 재래 산양의 체세포 핵이 식에 의한 복제 산자 생산에서 보다 효율성을 높이고 수태율을 향상시키기 위한 조건은 체내 성숙 난자를 수핵 난자로 이용하여 공란 산양과 발정이 동기화된(${\pm}0$) 자연 발정 수란 산양에 복제 수정란을 이식하는 것이 바람직한 것으로 생각된다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권10호
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• pp.1420-1428
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• 2009

In this study we cloned and characterized a novel lipid-accumulating gene, the oxidized low-density lipoprotein receptor 1 (OLR1), which is associated with lipogenesis. We analyzed the gene structure and detected the mRNA transcriptional expression levels in pig adipose tissues at different months of age (MA) and in different economic types (lean type and obese type) using real-time fluorescence quantitative PCR. OLR1 expression profile in different tissues of pig was analyzed. Finally, we studied the correlation between OLR1 and lipid metabolism related genes including peroxisome proliferator-activated $receptor{\gamma}2$ ($PPAR{\gamma}2$), fatty acid synthetase (FAS), triacylglycerol hydrolase (TGH), CAAT/enhancer binding protein $\alpha$ ($C/EBP{\alpha}$) and sterol regulatory element binding protein-1c (SREBP-1c). Results indicated that the OLR1 gene of the pig exhibited the highest homology with the cattle (84%), and the lowest with the mouse (27%). The signal peptide located from amino acid 38 to 60 and the domain from amino acid 144 to 256 were shared by the C-type lectin family. The expression level of OLR1 in pig lung was exceedingly higher than other tested tissues (p<0.01). In pig adipose tissue, the expression level of OLR1 mRNA increased significantly with growth (p<0.01). The expression level of OLR1 mRNA in obese-type pigs was significantly higher than that of lean-type pigs of the same monthly age (p<0.05). In adipose tissue, the expression of OLR1 correlated with $PPAR{\gamma}2$, FAS and SREBP-1c, but not TGH or C/EBP${\alpha}$. In conclusion, OLR1 was highly associated with fat deposition and its transcription, as suggested by high correlations, was possibly regulated by $PPAR{\gamma}2$ and SREBP-1c.

• Asian-Australasian Journal of Animal Sciences
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• 제20권2호
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• pp.184-193
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• 2007

In mammalian ovary, steroidogenic acute regulatory (StAR) protein mediates the true rate-limiting step of transport of cholesterol from outer to inner mitochondrial membrane. Appropriate expression of StAR gene represents an indispensable component of steroidogenesis and its regulation has been found to be species specific. However, limited information is available regarding StAR gene expression during estrous cycle in buffalo ovary. In the present study, expression, localization and hormonal regulation of StAR mRNA were analyzed by semi-quantitative RT-PCR in buffalo ovary and partial cDNA was cloned. Total RNA was isolated from whole follicles of different sizes, granulosa cells from different size follicles and postovulatory structures like corpus luteum and Corpus albicans. Semi-quantitative RT-PCR analyses showed StAR mRNA expression in the postovulatory structure, corpus luteum. No StAR mRNA was detected in total RNA isolated from whole follicles of different size including the preovulatory follicle (>9 mm in diameter). However, granulosa cells isolated from preovulatory follicles showed the moderate expression of StAR mRNA. To assess the hormonal regulation of StAR mRNA, primary culture of buffalo granulosa cells were treated with FSH (100 ng/ml) alone or along with IGF-I (100 ng/ml) for 12 to 18 h. The abundance of StAR mRNA increased in cells treated with FSH alone or FSH with IGF-I. However, effect of FSH with IGF-I on mRNA expression was found highly significant (p<0.01). In conclusion, differential expression of StAR messages was observed during estrous cycle in buffalo ovary. Also, there was a synergistic action of IGF-I on FSH stimulation of StAR gene.

• Asian-Australasian Journal of Animal Sciences
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• 제28권12호
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• pp.1721-1728
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• 2015

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (-420/+181) bovine NANOG 5'-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (-420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.23-31
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• 2011

Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제18권6호
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• pp.892-897
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• 2005

Macrophage colony-stimulating factor (M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocyte lineage. Total and 16 poly (A) mRNA of bovine M-CSF were isolated from healthy bovine peripheral mononuclear cells stimulated by phobol 12-myristste 13-acetate (TPA). The more compatible cultured mononuclear cells were 5${\times}$10/ml for RNA isolation. TPA-activated mononuclear cells increased the level of M-CSF-mRNA more than concanavalin A (Con A) and lipopolysaccharide (LPS). The optimal analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) for14 Macrophage colonystimulating factor (M-CSF) as a growth factor required for bovine M-CSF was denaturation at 94$^{\circ}C$ for 1 minute, annealing at 57$^{\circ}C$ for 1 minute, extension at 72$^{\circ}C$ for 1 minute for 30 cycles. The size of cDNA of bovine M-CSF by RT-PCR was 774 base pairs. A 774 base pairs cDNA encoding bovine M-CSF was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Ligated cDNA was transformed to competent cells and then plasmid isolation and digestion was performed. Molecular cloning and sequencing were performed for cDNA of bovine M-CSF. The size of cloned cDNA of bovine M-CSF was 774base pairs. The homology of base sequence and amino acid sequence was 88% and 86% compared with known human M-CSF, respectively. From a high degree of sequence similarity, the obtained cDNA of bovine M-CSF is thought be a specific gene of bovine M-CSF.

• 한국가축번식학회지
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• 제26권4호
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• pp.385-394
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• 2002

This study was constructed the correlations of the embryonic developmental rates and the frequency of chromosome aberration using ear-skin-fibroblast cell in nuclear transfer (NT) derived embryos. Karyoplast-oocyte complexes were fused and activated simultaneously, then cultured for seven days to assess development. The developmental rates of NT and in vitro fertilization (IVF) embryos were 55.4% vs 63.5%, 31.7% vs 33% and 13.4% vs 16.8% in 2 cell, 8 cell and blastocyst, respectively. Firstly, the frequency of chromosome aberrations were evaluated using fluorescent in situ hybridization (FISH) technique with porcine chromosome 1 submetacentric specific probe. Chromosome aberration was detected at day 3 on the embryo culture, the percentages of chromosomal aneuploidy in NT and IVF embryos at 4-cell stage were 40%, 31.3%, respectively. Secondly, embryonic fragmentation was evaluated at 4-cell stage embryo. Frequency of embryonic fragmentations was in 51.3% of NT, 61.3% of IVF, 28.9% of parthenogenetic activation at 4-cell stage. The proportion of fragmentation in NT embryos was higher than activation embryos. This result indicates that chromosomal abnormalities and embryonic fragments are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT related with lower implantation rate, increased abortion rate and production of abnormal fetuses.

• 한국가축번식학회지
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• 제25권4호
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• pp.381-388
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• 2001

본 연구는 돼지에서 성장호르몬과 결합되는 부위에 변이를 유도하여 결합력이 향상된 성장호르몬 결합단백질을 획득하기 위하여 수행되었다. 돼지의 지방으로부터 얻은 성장호르몬 수용체 RNA 내 성장호르몬 결합단백질 부분을 756 bp의 cDNA로 전향하고 클로닝한 후 site-directed mutagenesis 방법을 이용하여 26과 122번째 아미노산을 변이시켰다. 26번째 아미노산은 성장 호르몬과의 결합에 관련이 있다고 알려져 있는 돼지 성장호르몬 수용체 외막에 존재하는 다섯 군데의 N-linked glycosylation 부위와 가까이 위치한 부분이고, 122번째 아미노산은 소에서의 결합부위로 알려져 있다. 이렇게 변이를 유도한 성장호르몬 결합 단백질을 pET-32(c) 발현벡터에 삽입시키고 과발현시켰고 이를 정제하여 30 kDa의 변이를 유도한 성장호르몬 결합 단백질을 얻었다. 이러한 방법으로 성장호르몬 결합 단백질을 성장기에 있는 세포나 동물에 주입한다면 보다 향상된 성장을 볼 수 있을 것으로 사료된다.