• BMB Reports
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• v.33 no.2
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• pp.184-187
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• 2000

Prostaglandin $E_2$ ($PGE_2$) plays an important role in the regulation of various gastric functions, and the growth-inhibitory activities on tumor cells are studied in vitro and in vivo. Although the mechanisms have attracted many researchers in the past decade, the molecular mechanisms of cell cycle arrest, or induction of apoptosis by $PGE_2$, is unclear. We investigated the effects of $PGE_2$ on the growth of the human gastric carcinoma cell line SNU1 and genes that are regulated by $PGE_2$ and isolated them using differential display RT-PCR (DD RT-PCR). FACS analysis suggested that SNU1 cells were arrested at the G1 phase by $PGE_2$ treatment. This growth inhibitory effect was in a time- and dose-dependent manner. Treatment of SNU1 cells with $10\;{\mu}g/ml$ $PGE_2$, followed by DD RT-PCR analysis, revealed differently expressed bands patterns from the control. Among the differently expressed clones, we found an unidentified cDNA clone (HGP-27) overexpressed in $PGE_2$-treated cells. The full-length cDNA of HGP-27 was isolated using RACE, which consisted of a 30-nt 5'-noncoding region, a 891-nt ORF encoding the 296 amino acid protein, and a 738-nt 3'-noncoding region including a poly(a) signal. This gene was localized on the short arm of chromosome number 11. Using the Motif Finder program, a myb-DNA binding repeat signature was detected on the ORF region. The COOH-terminal half was shown to have similarity with the $NH_3$-terminal domain of thioredoxin (Trx). This relation between HGP-27 and Trx implied a potential role for HGP-27 in modulating the DNA binding function of a transcription factor, myb.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2005.05a
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• pp.239-242
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• 2005

본 연구는 성장 속도 및 서로 다른 육질 특성을 지닌 돼지 품종을 이용하여, 육질 및 성장에 관련된 유전자원을 확보하고, 이를 이용한 유전 육종의 기초 자료를 제공하기 위하여 수행하였다. Differential display (DD) RT-PCR 기법을 통해 돼지 품종 간 발현 차이를 보이는 유전자인 NADH dehydrogenase 1과 ATPase 6를 동정하였다. 동정된 유전자의 발현량 분석을 위한 RT-PCR 결과, 각 유전자의 발현량이 재래돼지에서 외래 품종 (랜드레이 스 및 요크셔)에 비해 2배 이상 높음을 확인 할 수 있었다 (p<0.01). 이러한 발현차이 유전자를 이용하여 육질과의 관련성 연구 및 유전자의 기능에 대한 연구가 지속되어야 할 것이다.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.74.1-74
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• 2003

Differential display (DD) of mRNA is a technique in which mRNA species expressed by a cell population are reverse transcribed and then amplified by many separate polymerase chain reactions (PCR). Using DD-RT-PCR we obtained many genes that expressed differentially in healthy and PVX-infected Nicotiana benthamima, using total RNAs extracted from healthy and PVX-infected N. benthamiana plants. Three hundred and twenty-five DNA fragments isolated from DD-RT-PCR were cloned and sequenced for further characterization. Several host genes including SKPI-like protein, heat shock transcription factor and Avr9/Cf-9 rapidly elicited protein were selected to obtain full-length open reading frame and to characterize their potential involvement in virus disease development and/or host's defense against virus infection employing PVX-based expression vector. Transcrips from wild-type and clones containing each selected gene were inoculated onto N. benthamiana Levels of virus replication were confirmedby RT-PCR and RNA blot analysis, Expression profiles and potential role(s) of selected genes upon PVX infection will be discussed.

• Journal of the korean academy of Pediatric Dentistry
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• v.32 no.1
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• pp.75-88
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• 2005

Ameloblastoma is the most commonly occurring odontogenic tumor in oral cavity. Although most are benign epithelial neoplasm, they are generally considered to be locally aggressive and destructive, exhibiting a high rate of recurrence. The biological behavior of this neoplasm is a slowly growing, locally invasive tumor without metastasis, therefore malignant neoplasm, changed its histological appearance to carcinoma or showed distant metastasis, is only defined clinically. In this study, we identified the differentially expressed genes(DEGs) in stages under benign or malignant ameloblastoma compared with normal patient using ordered differential display(ODD) reverse transcription(RT)-PCR and $GeneFishing^{TM}$ technology. ODD RT-PCR is rather effective when the investigation of samples containing very small amounts of total RNA must be accomplished. ODD RT-PCR used the means of amplification with anchored T-primer and adaptor specific primer. bearing definite two bases at their 3' ends and so this method could display differential 3'-expressed sequence taqs(ESTs) patterns without using full-length cDNAs. Compared with standard differential display, ODD RT-PCR is more simple and have enough sensitivity to search for molecular markers by comparing gene expression profiles, However, this method required much effort and skill to perform. $GeneFishing^{TM}$ modified from DD-PCR is an improved method for detecting differentially expressed genes in two or more related samples. This two step RT-PCR method uses a constant reverse primer(anchor ACP-T) to prime the RT reaction and arbitrary primer pairs(annealing control primers, ACPs) during PCR. Because of high annealing specificity of ACPs than ODD RT-PCR, the application of $GeneFishing^{TM}$ to DEG discovery generates reproducible, authentic, and long(100bp to 2kb) PCR products that are detectable on agarose gels. Consequently, various DEGs observed differential expression levels on agarose gels were isolated from normal, benign, and malignant tissues using these methods. The expression patterns of the some isolated DEGs through ODD RT-PCR and $GeneFishing^{TM}$ were confirmed by Northern blot analysis and RT-PCR. The results showed that these identified DEGs were implicated in ameloblastoma neoplasm processes. Therefore, the identified DEGs will be further studied in order to be applied in candidate selection for marker as an early diagnosis during ameloblastoma neoplasm processes.

• International Journal of Oral Biology
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• v.37 no.4
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• pp.167-173
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• 2012

This study investigated the genes involved in the differentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sorting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-polymerase chain reaction (DD-PCR) was performed to screen differentially expressed genes involved in the differentiation of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as ${\beta}$-glycerophosphate (${\beta}$-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induction. RT-PCR analysis revealed that dentin sialophosphoprotein (DSPP) and osteocalcin (OC) expression had increased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were differentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Furthermore, the characterization of these regulated genes during odontogenic induction will likely provide valuable new insights into the functions of odontoblasts.

• Archives of Pharmacal Research
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• v.28 no.2
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• pp.232-237
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• 2005

Mast cells play an important role in allergic inflammation by releasing their bioactive mediators. The function of mast cells is enhanced by stimulation because of the induction of specific genes and their products. While many inducible genes have been elucidated, we speculated that a significant number of genes remain to be identified. Thus, we applied differential display (dd) PCR to establish a profile of the induced genes in bone marrow-derived mast cells (BMMCs) after they were co-cultured with 3T3 fibroblasts. To date, 150 cDNA fragments from the connective-type mast cells (CTMCs) were amplified. Among them, thirty cDNA fragments were reamplified for cloning and sequencing. The ddPCR strategy revealed that serine proteases were the most abundant genes among the sequenced clones induced during the maturation. Additionally, unknown genes from the co-culture of BMMCs with 3T3 fibroblasts were identified. We confirmed their induction in the CTMCs by Northern blot analysis and RT-PCR. Characterization of these induced genes during the maturation processes will provide insight into the functions of mast cells.

• Molecules and Cells
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• v.23 no.3
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• pp.287-303
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• 2007

Expressed sequence tags (ESTs) and differentially expressed cDNAs from the self-fertilizing fish, Kryptolebias marmoratus were mined to develop alternative biomarkers for endocrine-disrupting chemicals (EDCs). 1,577 K. marmoratus cDNA clones were randomly sequenced from the 5'-end. These clones corresponded to 1,518 and 1,519 genes in medaka dbEST and zebrafish dbEST, respectively. Of the matched genes, 197 and 115 genes obtained Unigene IDs in medaka dbEST and zebrafish dbEST, respectively. Many of the annotated genes are potential biomarkers for environmental stresses. In a differential display reverse transcriptase-polymerase chain reaction (DD RT-PCR) study, 56 differential expressed genes were obtained from fish liver exposed to bisphenol A. Of these, 16 genes were identified after BLAST search to GenBank, and the annotated genes were mainly involved in catalytic activity and binding. The expression patterns of these 16 genes were validated by real-time RT-PCR of liver tissue from fish exposed to bisphenol A. Our findings suggest that expression of these 16 genes is modulated by endocrine disrupting chemicals, and therefore that they are potential biomarkers for environmental stress including EDCs exposure.

• International Journal of Oral Biology
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• v.37 no.2
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• pp.51-56
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• 2012

Tooth development involves bud, cap, bell and hard tissue formation stages, each of which is tightly controlled by regulatory molecules. The aim of this study was to identify genes that are differentially expressed during dental hard tissue differentiation. Sprague-Dawley rats at postnatal days 3, 6 and 9 were used in the analysis. Differential display RT-PCR (DD-PCR) was used to screen differentially expressed genes between the 2nd (root formation stage, during mineralization) and 3rd (cap stage, before mineralization) molar germs at postnatal day 9. The DNA detected in the 2nd molar germs showed homology to osteonectin only (GenBank accession no. NM_012656.1). The level of osteonectin mRNA expression was much higher in the 2nd molar germs than in the 3rd molar germs and was found to increase in a time-dependent manner from the early bell stage to the root formation stage in the 2nd molar germs. The pattern of osteonectin protein expression was consistent with these RT-PCR results. Osteonectin protein was found by immunofluorescent analysis to localize in odontoblasts and preodontoblasts rather than the dentin matrix itself. Further studies are needed to validate the involvement of osteonectin in mineralization and root formation.

• The Journal of the Korean Society for Microbiology
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• v.35 no.2
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• pp.149-157
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• 2000

Mycobacterium tuberculosis is capable of growing and survival within macrophage. The purpose of this study was to identify the genes regulated by infection of mycobacteria in human monocytic THP-1 cells. We used the differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) and nothern blot analysis to confirm the differentially expressed genes from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv, heat-killed Mycobacterium tuberculosis H37Rv and live Mycobacterium bovis BCG. Among many up or down-regulated clones, 27 clones were sequenced and compared with known genes on GenBank. Thirteen of over-expressed clones from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv were identical to human prothymosin alpha, eight were novel clones and six clones showed homology with Human ferritin H chain, Esherichia coli bgl, Mouse RNA-dependent EIF-2 alpha kinase, E. coli htrL, Hyaluronan receptor and T cell receptor. Our result suggests that Mycobacterium tuberculosis might regulate prothymosin alpha gene transcription in monocytic THP-1 cell.

• Molecular & Cellular Toxicology
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• v.4 no.4
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• pp.293-299
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• 2008

To screen the differentially expressed genes in cadmuim-exposed marine medaka fish (Oryzias javanicus), a candidate marine test fish for ecological toxicity, the differential display polymerase chain reaction (DD-PCR) was carried out, since the genome-wide gene expression data are not available in this fish species yet. A total of 35 clones were isolated from cadmium-exposed fish and their nucleotide sequences were analyzed. The differentially expressed gene candidates were categorized to response to stimulus (3); ion binding (3); DNA binding (1); protein binding (6); carbohydrate binding (1); metabolic process (4); biological regulation (3); cellular process (2); protein synthesis (2); catalytic activity (2); sense of sight (1); immune (1); neurohormone (1); signaling activity (1); electron carrier activity (1) and others (3). For real-time quantitative RT-PCR, we selected catalase, glucose-6-phosphate dehydrogenase, heat shock protein 70, and metallothionein and confirmed that cadmium exposure enhanced induction of these four genes.