• 한국가금학회:학술대회논문집
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• 한국가금학회 1999년도 제16차 정기총회및학술발표회
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• pp.60-61
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• 1999

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1383-1391
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• 2016

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

• 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.

• Animal cells and systems
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• 제15권3호
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• pp.227-233
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• 2011

The Nramp1/Slc11a1 locus encodes a proton-coupled divalent cation transporter, expressed in late endosomes/lysosomes of macrophages, that constitutes a component of the innate immune response to combat intracellular pathogens and it was shown to play an important role in regulating inherent immunity. The previously identified Z-DNA forming polymorphic repeat(GT)n in the promoter region of the human Nramp1 gene does act as a functional polymorphism influencing gene expression. Research has shown that INF-${\gamma}$, TNF-${\alpha}$, IL-$1{\beta}$ and bacteria LPS increase the level of Nramp1 expression. However, the molecular mechanism for Nramp1 gene regulation is unclear. In this research, bovine Nramp1 5'-flanking region (-1748~+769) was cloned and analyzed by bioinformatics. Then to find the core promoter and the cis-acting elements, deletion analysis of promoter was performed using a set of luciferase reporter gene constructs containing successive deletions of the bovine Nramp1 5'-flanking regions. Promoter activity analysis by the dual luciferase reporter assay system showed that the core promoter of Nramp1 was located at +58~-89 bp. Some positive regulatory elements are located at -89~-205 bp and -278~-1495 bp. And the repressor elements were in region -205~-278 bp, intron1 and -1495~-1748 bp. LPS-responsive regions were located at -1495~-1748 bp and -278~-205 bp. The present study provides an initial effort to explore the molecular mechanism of transcriptional activation of the bovine Nramp1 gene and should facilitate further studies to decode the complex regulatory process and for molecular breeding for disease resistance in bovines.

• Journal of Plant Biotechnology
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• 제40권1호
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• pp.27-36
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• 2013

We isolated and functionally characterized a Dendrobium Actin1 (DmACT1) promoter that drives strong gene expression in the orchid genus Dendrobium. A genomic fragment containing the region 3227 bp upstream of the coding region of DmACT1 was obtained by inverse PCR. Detailed comparison of the full-length cDNA and genomic sequences revealed that DmACT1 has a 1374 bp first intron in the 5' UTR. However, the 5' flanking sequences upstream of the coding region showed no obvious sequence similarities compared to those of known promoters, including plant actin promoters. Serial deletion constructs of the 5' flanking region from the translation initiation codon were fused to the coding sequence of a GUS/luciferase fusion reporter to identify the regulatory elements necessary for promoter activity. Transient assays in the flowers of Dendrobium revealed that the 5' UTR-intron greatly enhanced promoter activity. Moreover, the DmACT1 promoter with its 5' UTR-intron yielded approximately 10-fold higher reporter activity than the rice Act1 promoter-intron. Our data suggest that the DmACT1 promoter with its 5' UTR-intron is a useful tool for strong expression of transgenes in Dendrobium orchids.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2001년도 공동 춘계학술대회
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• pp.89.2-89
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• 2001

• 농업과학연구
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• 제27권1호
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• pp.33-38
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• 2000

본 연구는 한우와 Holstein종의 $\kappa$ -CN 유전자 발현조절부위, 즉 $\kappa$ -CN gene 5'-flanking region에 대한 특성을 구명하기 위하여 specific primer를 이용하여 PCR(polymerase chain reaction) 방법으로 분석하였다. PCR 증폭 결과, 349bp 크기의 증폭산물을 얻었으며 한우와 Holstein종의 $\kappa$ -CN 유전자의 발현조절부위의 염기서열 분석결과에 있어서는 -82bp에서 -431bp까지 분석되었으며, 종간의 염기서열 비교에서 -386bp에서는 Holstein종에서는 T 염기가 한우에서는 C 염기로 치환되었으며, -241bp와 -192bp에서는 Holstein종에서는 T 및 C 염가가 존재하였으나, 한우에서는 결실되었다. 또한, -183bp에서는 한우에서는 T 염기가 존재하였으나, Holstein종에서는 염기가 결실되어 있음을 알 수 있었다. 또한 한우와 Holstein 종간의 상동성 분석에 있어서는 한우 $\kappa$ -CN 유전자 발현조절부위의 염기서열이 Holstein종과 98.9% 의 상동성을 보였다.

• 한국발생생물학회지:발생과생식
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• 제7권2호
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• pp.119-126
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• 2003

본 연구실에서는 이전의 실험에서 suppression subtractive hybridization(SSH)을 통하여 생쥐의 생후 1일자 난소와 5일자 난소에서 차이 나게 발현하는 유전자들의 목록을 얻었고 그 중에서 MT transposon-like element, clone MTi7(MTi7)이 성장하는 난포에서 더 높게 발현한다는 것을 알아냈다(Park et al., 2002). In situ hybridization과 RNA interference를 이용한 연구결과, MTi7은 난자에서 특이 적으로 발현하는 유전자로 특히 난자성숙에 관여하는 것으로 관찰되었다(Park et at., 2003). 그러나 현재까지 MTi7의 염기서열은 생쥐에서만 알려져 있다. 따라서 본 연구는 두 부분으로 나누어서 첫째, MTi7이 다른 포유류에도 존재하는지 알아보기 위해 소,돼지, 흰쥐 그리고 사람 등 각기 다른 네 종의 난소 cDNA를 사용하여 새로운 MTi7을 분리하고자 하였으며, 둘째, 생쥐의 MTi7이 transposon의 특징을 갖고 있어 다른 유전자에 삽입되어 있는지를 알아보기 위해 inverse PCR을 시행하여 MTi7주변의 유전자가 있는지를 조사하였다. 네 종의 난소 cDNA를 사용하여 생쥐의 MTi7과 매우 유사한 염기서열을 갖고 있음을 알았다(87%∼98%). Inverse PCR 결과, 생쥐의 MTi7은 beta-carotene 15, 15'-monooxygenase(Bcdo) 유전자 혹은 serine protease inhibitor, Kunitz type I(Spint 1) 유전자에 삽입되어 있음을 알 수 있었다. 본 연구의 결과로 여러 포유류의 MTi7 sequence를 알아내고, 또한 생쥐의 MTi7이 삽입되어 있는 유전자를 알아냄으로써 머지 않은 장래에 난자형성 및 난포형성과정에 있어서 MTi7의 역할을 밝혀 낼 수 있을 것으로 기대된다.

• 약학회지
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• 제43권5호
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• pp.584-590
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• 1999

Bioactivity-directed fractionation from the seeds of Annona cherimolia resulted in the isolation of two known cytotoxic compounds : corrosolin (1) and compound-2 (2). The structures of these compounds were characterized on the basis of chemical and spectral data. Corrosolin has a relative stereochemical relationship of threo/trans/threo for the mono-tetrahydrofuran (THF) ring with two flanking hydroxyls, from C-15 to C-20, which is the annonacin type. Compound-2 has a relative stereochemical relationship of threo/trans/threo/trans/threo for the adjacent bis-THF ring with two flanking hydroxyls, such as in the asimicin type. The absolute configurations of carbinol carbons in corrosolin in corrosolin were determined as 10R, 15R, and 20R by analysis of its Mosher ester derivatives. Corrosolin and compound-2 are known, but are first isolated in this plant.

• Reproductive and Developmental Biology
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• 제32권1호
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• pp.21-25
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• 2008

Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal and differentiation into a variety of cell types. They represent an attractive source of cells for gene and cell therapy. The purpose of this study is to direct the specific expression of the DsRed reporter gene in $Sca-1^+$ BMMSCs differentiated into a cardiomyogenic lineage. We constructed the prMLC-2v-DsRed vector expressing DsRed under the control of the 309 tp fragment of the rat MLC-2v 5'-flanking region. The specific expression of the DsRed reporter gene under the transcriptional control of the 309 bp fragment of the rat MLC-2v promoter was tested in 5-azacytidine healed-$Sca-1^+$ BMMSCs over 2 weeks after the prMLC-2v-DsRed transfection. The prMLC-2v-DsRed was specifically expressed in the $Sca-1^+$ BMMSCs with cardiomyogenic lineage differentiation and it demonstrates that the 309 bp sequences of the rat MLC-2v 5'-flanking region is sufficient to confer cardiac specific expression on a DsRed reporter gene. The cardiac-specific promoter-driven reporter vector provides an important tool for the study of stem cell differentiation and cell replacement therapy in ischemic cardiomyopathy.