• Reproductive and Developmental Biology
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• 제33권4호
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• pp.249-255
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• 2009

The miniature pig is considered to be a better organ donor breed for xenotransplantation than other pig breeds because the size of the organs of the miniature pig is similar to that of humans. In this study, we aimed at identifying differentially expressed genes in the miniature pig ovary during pregnancy. For this, we used the miniature pig ovary model, annealing control primer-based reverse transcription polymerase chain reaction (PCR), quantitative real-time PCR (qRT-PCR), and northern blotting analysis. We identified 13 genes showing differential expression on the based of pregnancy status and validated 8 genes using qRT-PCR. We also sequenced the full-length cDNA of ephrin receptor A4 (EphA4), which had a significant difference in expression level, and validated it by northern blotting. These genes may provide a better understanding of the cellular and molecular mechanisms during pregnancy in miniature pig ovary.

• 원예과학기술지
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• 제18권4호
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• pp.513-517
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• 2000

우리나라 난 재배 농가에서 많이 재배하고 있는 호접란 대만 수입묘의 바이러스 감염 정도를 검정하기 위하여 플라스크묘 상태의 실생 번식 식물체를 공시하여 역전사 중합효소 연쇄반응(RT-PCR) 기술에 의해 CymMV와 ORSV의 감염 여부를 검정하였다. 호접란 식물체의 잎에서 조즙액을 추출하여 RT-PCR을 위한 total RNA로 사용하였다. $42^{\circ}C$에서 45분간 반응시켜 cDNA를 합성하였으며, $96^{\circ}C$에서 2분간 template를 예비 변성시킨 후, $96^{\circ}C$에서 30초간 template 변성, $60^{\circ}C$에서 30초간 primer 부착 및 $72^{\circ}C$에서 1분간 DNA 합성을 1cycle로 하여 총 36cycle을 반응시키고, $72^{\circ}C$에서 10분간 안정화하는 조건으로 one-step RT-PCR을 수행하였다. 바이러스 검정 결과 정도의 차이는 있으나, 40개 시료 모두 CymMV에 감염되어 있었으며, ORSV에 감염된 식물체는 없었다.

• 한국식품위생안전성학회지
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• 제29권1호
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• pp.31-39
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• 2014

본 연구에서는 FCV 현탁액에 물리, 화학적 위생처리 후 복합효소처리라는 전처리과정을 적용한 뒤 real-time RT-PCR법을 이용하여 살균효능을 분석하였다. RT-PCR 이전에 $37^{\circ}C$에서 30분 동안 PK와 RNase A를 처리함으로써 UV, 열, 염소, 에탄올, 과초산계열 제품에 의해 불활성화 된 바이러스들은 음성 결과를 나타내었고, real-time RTP-CR법을 통해 살균 효능을 정량분석한 결과, 복합효소처리를 했을 경우 무처리구보다 더 높은 살균 효능을 보이는 것을 확인할 수 있었다. 이로써 Nuanualsuwan S. 등의 선행연구에서와 같이 PK와 RNase A로 전처리하는 단계를 통하여 물리, 화학적 위생처리에 의해 손상되지 않은 바이러스가 RT-PCR법에 의해 증폭되는 것을 방지함으로써 Real-time PCR법에 대한 검출 감도를 높일 수 있음을 확인하였다. 또한, FCV를 검출하기 위해 사용된 RT-PCR과 real-time RT-PCR 두 방법 중에서도 real-time RT-PCR법이 가장 신속하면서도 민감도 높은 결과로 도출되었다. 따라서, 유전자 분석 이전에 복합효소처리는 물리, 화학적 위생처리에 의해 불활성화 된 바이러스의 RNA가 transcription 또는 증폭되는 것을 방지하기 위한 수단으로 real-time RT-PCR법과 결합됨으로써 노로바이러스를 비롯한 식중독 바이러스를 검출하는데 효과적으로 적용될 것으로 판단된다. 또한 식품현장에서 전기영동 과정없이 신속하게 살아있는 바이러스만을 수치적으로 정량화함으로써 식품안전에도 기여할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제22권5호
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• pp.622-627
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• 2012

Mast cells and basophils are important effector cells in immunoglobulin-E (IgE)-mediated allergic reactions. Using the human basophilic KU812F cells, we assessed the inhibitory effects of 6-methoxyluteolin, isolated from Chrysanthemum zawadskii, in the $Fc{\varepsilon}RI$-mediated allergic reaction. We determined that 6-methoxyluteolin inhibited anti-$Fc{\varepsilon}RI$ ${\alpha}$ chain antibody (CRA-1)-induced histamine release, as well as elevation of intracellular calcium concentration $[Ca^{2+}]_i$ in a dose-dependent manner. Moreover, the inhibitory effects of 6-methoxyluteolin on the cell surface expression and the mRNA level of the $Fc{\varepsilon}RI$ ${\alpha}$ chain were determined by flow cytometric analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Therefore, these results show that 6-methoxyluteolin is a potent inhibitor of histamine release and calcium influx via down-regulation of the $Fc{\varepsilon}RI$ ${\alpha}$ chain.

• 한국가금학회:학술대회논문집
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• 한국가금학회 1999년도 제16차 정기총회및학술발표회
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• pp.34-50
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• 1999

A cDNA encoding chicken interferon-gamma (chIFN-${\gamma}$) was amplified from P34, a CD4$^{+}$ T-cell hybridoma by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pUC18. THe sequences of cloned PCR products were determined to confirm the correct cloning. Using this cDNA as probe, chicken genomic library from White Leghorn spleen was screened. Phage clones harboring chicken interferon-gamma (chIFN-${\gamma}$) were isolated and their genomic structure elucidated. The chIFN-${\gamma}$ contains 4 exons and 3 introns spanning over 14 kb, and follows the GT/AG rule for correct splicing at the exon/intron boundaries. The four exons encode 41, 26, 57 and 40 amino acids, respectively, suggesting that the overall structure of IFN-${\gamma}$ is evolutionairly conserved in mammalian and avian species. The 5’-untranslated region and signal sequences are located in exon 1. Several AT-rich sequences located in the fourth exon may indicate a role in mRNA turnover. The 5’-flanking region contains sequences homologous to the potential binding sites for the mammalian transcription factors, activator protein-1(AP-1) activator protein-2(AP-2) cAMP-response element binding protein(CREB), activating transcription factor(ATF), GATA-binding fator(GATA), upstream stimulating factor(USF), This suggests that the mechanisms underlying transcriptional regulation of chicken and mammalian IFN-${\gamma}$ genes may be similar.r.

• 식물병연구
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• 제7권2호
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• pp.80-85
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• 2001

이 연구는 고랭지 나리에서 발생하는 바이러스의 병징, 종류 및 계통별 방병률을 조사 분석하고 효과적인 검정방법을 개발하고자 수행하였다. 고랭지 나리에서 발생하는 바이러스의 병징은 모자이크, 축엽, 퇴록반점, 줄무의, 라인패턴을 나타내었으며, 증상별 분포는 모자이크가 43.8%, 축엽이 29.2%, 퇴록반점이 10.9%였다. 바이러스 종류별로는 Lily symptomless virus(LSV), Cucumber mosaic virus(CMV), Lily mottle virus(LMoV) 등 6가지 바이러스가 전자현미경으로 검정되었다. 지역별로는 강릉(왕산)이 대관령보다 바이러스 이병률이 높았으며, 계통별 바이러스 이병률은 오리엔탈 계통(카사블랑카, 마르코폴로)이 아시아틱 계통(솔레미오, 플라토)보다 2~4배 높았다. 바이러스 진단방법으로는 기존의 PT-PCR보다 개선된 one-step RT-PCR 검정이 시간을 줄이면서 민감도가 뛰어나 가장 효과적이었다.

• 대한한의학회지
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• 제23권2호
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• pp.190-197
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• 2002

Objectives : This study was performed to examine the effect of Yukmigiwhang-tang kamibang, a mixture of oriental herbal extracts, on the transcription of bone fonnation genes, BMP4 (bone morphogenetic protein 4) and TG2 (transglutaminase-2). Methods : Bone-related cells, MG-63 (human male osteosarcoma), HOS-TE85 (human female osteosarcoma), and KG-l (bone marrow) were cultured with portions of Yukmigiwhang-tang kamibang and the transcription activities of bone-related genes, BMP4 (bone morphogenetic protein 4) and TG2 (transglutaminase-2), were determined by Reverse-Transcription Polymerase Chain Reaction (RT-PCR). Results : Transcription of BMP4 gene in HOS-TE85 cell increased up to 40% at 0.3% (v/v) of Yukmigiwhang- tang kamibang extract and that of TG2 gene in MG-63 cells also increased up to 40% at 0.3-0.4% of the same extract. Although it was less significant when compared to those in other cells, the transcription of BMP4 gene in KG-l cells also increased up to 10 to 25%. Conclusions : These results clearly demonstrated that Yukmigiwhang-tang kamibang have an effect on transcription activity of bone-related genes, TG2 and BMP4, suggesting that it may play an important role in bone formation.

• The Plant Pathology Journal
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• 제22권2호
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• pp.131-138
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• 2006

Two strains of Cucumber mosaic virus (CMV) isolated in Kuwait were confirmed their infectivity based on symptomatology and host range on different cultivars of tomato (Lycopersicon esculentum), tobacco(Nicotiana tabacum L.) and squash (Cucurbita pepo). The pattern of symptoms differed for the two CMV strains in tomato and tobacco, showing severe stunting and mosaic symptoms with one strain designated KU2, and almost symptomless with the other strain designated KU1. A satellite RNA 5 (sat-RNA) was found to be associated with the KU1 strain and was characterized as a benign viral satellite RNA. Using reverse transcription and polymerase chain reaction (RT-PCR) with sat-RNA specific primers, an amplified PCR product of about 160bp was determined and analyzed by gel electrophoresis. This naturally occurring benign viral satellite RNA was successfully used as a biological control agent to protect tomato plants against the severe KU2 strain. Tomato plants grown in plant-growth chambers, were preinoculated with KU1 containing the benign viral satellite and then challenge inoculated with the severe KU2 strain at different time intervals. All plants challenged three weeks after preinoculation showed nearly complete protection from subsequent infection by the severe strain. This biological control technology using plant viruses was found protective and could be successfully established sooner after the preinoculation.

• Parasites, Hosts and Diseases
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• 제37권4호
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• pp.285-287
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• 1999

Changes in the expression level of splenocyte $IFN-{\gamma}$mRNA of Sprague-Dawley (SD) rats infected with Paragonimus westermani were analyzed by competitive reverse transcription-polymerase chain reaction (RT-PCR) followed by southern blot. The template RNA was extracted from the splenocytes of rats infected with 20 metacercariae of P. westermani. The products of competitive RT-PCR were subjected to southern blot and enhanced chemiluminescence (ECL), and analyzed with a densitometer. In comparison with that of uninfected control rat splenocytes (value of 1), the levels of mRNA expression of $IFN-{\gamma}$had changed to 0.747 at 1 week post infection (PI), 0.00175 at 2 week PI, 0.0217 at 3 week PI, 0.194 at 4 week PI and then to 0.537 at 5 week PI. The level at 7 week PI had returned to 1.25, comparable with that of uninfected rats. These results show that, when infected with p. westermani, the levels of $IFN-{\gamma}$ mRNA of SD rat splenocytes were remarkably reduced by more than 500 times at 2 week PI and restored to normal level at 7 week PI.

• 대한바이러스학회지
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• 제27권2호
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• pp.169-176
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• 1997

In this study, the feasibility of identification and genotypic differentiation of enteroviruses was investigated by using nested reverse transcription-polymerase chain reaction (nested RT-PCR), single-stranded conformation polymorphism (SSCP), and restriction fragment length polymorphism (RFLP) techniques. Two hundred seventy-four clinical samples were assayed by both nested RT-PCR and tube culture method using MRC-5 and MK cells; 58 (86.6%) out of 67 enterovirus culture-positive samples contained enteroviral RNA. In addition, 114 (55.1%) of 207 samples from patients with suspected enteroviral CNS disease with negative viral cultures were positive by the nested RT-PCR. The nested RT-PCR products were genotyped by the SSCP method and the results were compared with serotypes. We could differentiate 6 subtypes, 3 of which are similar to coxsackievirus B3, B5, echovirus 11, plus 3 other subtypes. RFLP cleaved with Sty I, Bgl I, and Xmn I yielded characteristic patterns for each laboratory strains. This study demonstrates the usefulness of the RT-PCR for the rapid diagnosis of enterovirus infection and the potentials of the SSCP method for differentiation of enterovirus strains.