• 생명과학회지
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• 제14권5호
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• pp.732-740
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• 2004

MCM 단백질의 암세포내에서 과발현 되는 원인을 규명하기 위하여 mcm7, 2 promoter영역의 전사인자 결합 부위의 역할을 조사하였다. 암세포에서 promoter 활성은 정상세포보다 8배정도 높은 활성을 나타내 특유 전사인자의 존재를 시사하였다. Promoter영 역 에는 E2F 결합부위 와 Myc/Max/Mad단백질 결합 부위로 알려진 I-box가 존재한다. 이들 각각의 변이체 promoter를 작성하여 암세포와 정상세포에서 luciferase reporter 활성을 측정하는 것으로 각 영역의 역할을 검토하였다. 그 결과 정상세포 내에서는 강한 repressor존재 또는 활성을 negative로 조절 할 수 있는 complex의 존재로 promoter활성이 조절되는 반면 암세포 내에서는 I-box에 결합하여 promoter를 활성화시키는 특유 단백질의 존재가 시사되었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제39권1호
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• pp.22-28
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• 2019

We previously isolated 9 clones that show stronger signal compared to Bombyx mori cytoplasmic actin gene (BmA3) by using a dot blot hybridization. In this study, we focused on one clone among these clones which has high amino acid similarity with ${\beta}$-tubulin gene of B. mori. This clone was ubiquitously expressed in all tissues and developmental stage of B. mori. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-750/-1) in the 5'-flanking region of ${\beta}$-tubulin gene, which has about 47 fold more intensive promoter activity than BmA3 promoter. Moreover, the ${\beta}$-tubulin promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that ${\beta}$-tubulin promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6557-6562
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• 2013

Objective: Specific promoters could improve efficiency and ensure the safety of gene therapy. The aim of our study was to screen examples for lung cancer. Methods: The firefly luciferase gene was used as a reporter, and promoters based on serum markers of lung cancer were cloned. The activity and specificity of seven promoters, comprising CEACAM5 (carcinoembryonic antigen, CEA), GRP (Gastrin-Releasing Peptide), KRT19 (cytokeratin 19, KRT), SFTPB (surfactant protein B, SP-B), SERPINB3 (Squamous Cell Carcinoma Antigen, SCCA), SELP (Selectin P, Granule Membrane Protein 140kDa, Antigen CD62, GMP) and DKK1 (Dickkopf-1) promoters were compared in lung cancer cells to obtain cancer-specific examples with strong activity. Results: The CEACAM5, DKK1, GRP, SELP, KRT19, SERPINB3 and SFTPB promoters were cloned. Furthermore, we successfully constructed recombinant vector pGL-CEACAM5 (DKK1, GRP, SELP, KRT19, SERPINB3 and SFTPB) contained the target gene. After cells were transfectedwith recombinant plasmids, we found that the order of promoter activity from high to low was SERPINB3, DKK1, SFTPB, KRT19, CEACAM5, SELP and GRP and the order for promoters regarding specificity and high potential were SERPINB3, DKK1, SELP, SFTPB, CEACAM5, KRT19 and GRP. Conclusion: The approach adopted is feasible to screen for new tumour specific promoters with biomarkers. In addition, the screened lung-specific promoters might have potential for use in lung cancer targeted gene therapy research.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.81-81
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• 1997

We previously demonstrated an enhancer-like positive regulatory element within a 259-bp sequence (-2352 to-2094 bp) of the human CYP1A2 gene in HepG2 cells. Three protein binding sites were identified by DNase I footprint analyses within the 259-bp sequence: protected region A PRA ( -2283 to-2243 bp), PRB (-2218 to-2187 bp), and PRC (-2124 to-2098 bp) (I. Chung and E. Bresnick, Mol. Pharmacol. 47, 677-685, 1995). In the present study, the functional significance of those protected regions was examined. Transfection experiments with deletion and substitution mutants defined the PRB and PRC as containing positive and negative regulatory elements, respectively. Human breast carcinoma MCF-7 cells were cotransfected with a hepatocyte nuclear factor-1 (HNF-1) expression vector and CYP1A2 promoter-or thymidine kinase promoter-luciferase remoter gene constructs. HNF-1, which contributes to the liver specificity of genes, enhanced reporter gene activity in a PRC sequence-dependent manner. These results suggested that PRC could exist bound to a repressor which was displaceable by other transcription factors such as HNF-1. Results obtained by transfection of HepG2 hepatoma cells with various PRB substitution mutant-luciferase gene fusion constructs indicated that the entire sequence of PRB was necessary for promoter activity. Consequently, the regulation of CYP1A3 expression is very complex, requiring a number of both positive and negative regulatory factors.

• Macromolecular Research
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• 제14권3호
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• pp.348-353
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• 2006

For enhancing the gene delivery efficiency of polyplexes, a new formulation was developed using PEI conjugated Pluronic F127 copolymer as an effective additive. Low molecular weight, branched polyethylenimine Mw 600 (LMW BPEI 600) was conjugated to the terminal end of Pluronic F127. The PEI-modified Pluronic copolymers formed a micellar structure in aqueous solution, similar to that of unmodified Pluronic copolymer. PEI modification of Pluronic copolymer increased the size of micelles while concomitantly raising the critical micelle concentration (CMC). The PEI-modified Pluronic copolymer was used as a micellar additive to enhance the gene transfection efficiency of pre-formulated polyelectrolyte complex nanoparticles composed of luciferase plasmid DNA and branched PEI Mw 25k (BPEI 25k) or polylysine Mw 39k (PLL 39k). The luciferase gene expression levels were significantly enhanced by the addition of the BPEI-modified Pluronic copolymer for the two formulations of BPEl and PLL polyplexes. The results indicated that the BPEI-modified Pluronic copolymer micelles ionically interacted on the surface of DNA/BPEI (PLL) polyplexes which might facilitate cellular uptake process.

• Asian-Australasian Journal of Animal Sciences
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• 제24권11호
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• pp.1529-1540
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• 2011

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

• 대한바이러스학회지
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• 제30권1호
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• pp.39-49
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• 2000

The genome of Hantaan virus, the prototype of the hantavirus genus, is composed of three segmented, single stranded negative sense RNA genome. The 5' and 3' termini of the Hantaan virus RNA genome contain noncoding regions (NCRs) that are highly conserved and complementary to form panhandle structures. There are some reports that these NCRs seems to control gene expression and viral replication in influenza virus and vesicular stomatitis virus. In this study, we examined whether NCRs in Hantaan virus playa role in expression of the viral nucleocapsid protein (Np) and foreign (luciferase) gene. The 5' and/or 3' NCR-deleted mutants were constructed and analysed. The Np expression of 5' NCR-deleted clone was similar to that of the clone containing full S genome. In the case of 3' NCR-deleted clone, it showed 40% reduction. To investigate the role of NCR in foreign gene expression, the clones which are replaced ORF of Hantaan viral Np gene with that of luciferase gene were constructed. The results were similar to those of the experiments using Np gene. These results suggest that 3' NCR is more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in protein expression, several clones with a deleted part of 3' NCR were constructed and analyzed. The deletion of the conserved region in 3' NCR showed $20{\sim}30%$ decrease in Np expression. However there were no change in luciferase activities between clones with or without non-conserved region of 3' NCR. These results suggest that the 3' NCR of Hantaan virus S genome, especially conserved region in 3' NCR, plays an important role in the expression of Hantaan viral Np and foreign genes.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
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• pp.114-114
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• 2001

Since hypoxia-inducible factor-lalpha (HIF-lalpha) and the arylhydrocarbon receptor (AhR) shared the AhR nuclear translocator (Arnt) for hypoxia- and AhR-mediated signaling, respectively, it was possible to establish the hypothesis that hypoxia could regulate Cyplal expression. In order to understand the mechanism of Cyplal gene expression, we demonstrated here that hypoxic agents such as cobalt chloride, desferrioxarnine, and picolinic acid reduced the TCDD induced Cyplal promoter activity based on the determination of luciferase activity in Hepa I cells transfected with pmCypla1-Luc. Also cobalt chloride inhibited the TCDD stimulated Cyplal mRNA level as well as EROD activities in both Hepa I and MCF-7 cells. Hypoxic agents such as cobalt chloride, picolinic acid, and desferrioxamine showed inhibition of luciferase activity that was induced by lnM TCDD treatment with dose dependent manner. Concomitant treatment of 150${\mu}$M ferrous sulfate with 1∼100${\mu}$M desferrioxarnine or 1∼100${\mu}$M picolinic acid recovered from the hypoxic agents-inhibited luciferase activity that was stimulated by TCDD. Reciprocally, the hypoxic agents down regulated TCDD induced Cyplal mRNA level and CYP1A1 enzyme activity in Hepa I cells.

• 한국환경성돌연변이발암원학회지
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• 제23권2호
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• pp.45-50
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• 2003

Biological activities of PAHs are not known although PAHs are considered as carcinogens. Recent industrial society has human widely exposed to PAHs (polynuclear aromatic hydrocarbons) that are comming from the incomplete combustion of organic material as wider spread environmental contaminants. Our laboratory have been studied the effect of PAHs in the human breast cancer MCF-7 cells. In this study, we examined the human breast cancer MCF-7 cells as a new system to evaluate bioactivity of PAHs. We have selected 13 PAHs to examine bioassay using CYP1A1-luciferase reporter gene expression system where CYP1A1 1.6 Kb 5flanking region DNA was cloned in front of luciferase reporter gene and this plasmid was transfected into MCF-7 cells transiently. This cells then used for the study to observe the effect of PAHs. We demonstrated that PAHs induced the CYP1A1 promoter, CYP1A1 mRNA and 7-ethoxyresolufin O-deethylase (EROD) activities in a concentration-dependant manner. None of PAHs that we have tested showed stronger stimulatory effect on CYP1 gene expression than TCDD. Benz(a)anthracene and benzo(b)fluoranthene were weak responders to CYP1A1 promoter activity stimulation, CYP1A1 mRNA and EROD induction in MCF-7 cells and these chemicals seemed to respond less either CYP1A1 mRNA or EROD than CYP1A1 promoter activity. Benzo(k)fluoranthene, chrysene, and dibenzo(a, h)anthracene showed strong response to CYP1A1 promoter activity stimulation, CYP1A1 mRNA increase and also EROD induction in MCF-7 cells. Results of dose response study suggested that two strong responding PAHs, such as benzo(k)fluoranthene and dibenzo(a, h)anthracene might be mediated through Aryl hydrocarbon receptors system in MCF-7 cells.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.11-15
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• 2008

The expression of neuronal nitric oxide synthase (nNOS) is regulated by various spliced first exons (exon 1a-1i), sharing differentially common exon 2 in diverse human tissues. The highly complex structure and regulation of human nNOS gene gave limitations of information for the precise mechanism of nNOS regulation. In the present study, we report that the repeats of polymorphic dinucleotides $(GT)^nA(TG)^n$ repeats located in just upstream to the exon 1f in human nNOS gene play suppressive role in transcription, as shown in the characteristics of Z-DNA motif in other genes. In neuronal and trophoblast cells transfected transiently with luciferase construct without dinucleotide repeats at the 5'-flanking region of exon 1f in nNOS gene, the luciferase activity was increased markedly. However, the presence of the dinucleotide repeats dramatically suppressed the luciferase activity to the basal level, and which was dependent on the length of $(GT)^n$ and $(TG)^n$ repeats. More importantly, we found the polymorphisms in the length of dinucleotide repeats in human. Furthermore, we show for the first time here that there is a significant association of the lengths of polymorphic dinucleotide $(GT)^n$ and $(TG)^n$ repeats with the risk of schizophrenia.