• 한국잠사곤충학회지
• /
• 제52권1호
• /
• pp.39-44
• /
• 2014

효율적인 형질전환 누에 시스템 구축을 위해서는 새로운 전이인자의 개발과 함께 선발을 위한 마커 유전자 및 transposase 발현을 효과적으로 조절할 수 있는 다양한 유전자 프로모터 개발이 필수적이다. 이와 관련하여 선행연구를 통해 누에 후부실샘으로부터 고발현하는 RNA binding protein-1 homologue(RBP-1) 유전자를 선발한 바 있다. 본 연구에서는 RBP-1유전자의 누에 발육시기별 및 유충 조직별 발현양상을 Northen blot hybridization 방법으로 분석한 결과, RBP-1 유전자는 유충기로부터 번데기 후기까지의 전기간에 걸쳐 발현하였으며, 두부, 표피, 중장, 지방체 및 견사선 등 실험한 모든 유충 조직에서 고발현 하는 것으로 관찰되었다. 또한, 누에 게놈 유전자은행을 제작한 후 RBP-1 cDNA 유전자를 탐침으로 5'-UTR 영역을 클로닝하고 luciferase assay 방법으로 RBP-1 유전자 프로모터의 활성을 분석하였다. 실험 결과, RBP-1 cDNA를 탐침으로 RBP-1 유전자 ORF와 5'-UTR이 포함된 약 1,660 bp 영역의 게놈 유전자를 클로닝하였다. RBP-1 유전자 프로모터 활성검정을 위해 전사 개시점(+ 30)으로부터 상류의 -740 bp 영역을 PCR로 분리한 후 pGL3 basic vector에 도입하여 luciferase 활성 측정을 위한 전이벡터, pGL-RBP1를 제작하였다. 제작된 pGL-RBP1는 곤충 세포주(Sf9)에 transfection 한 후 luciferase 발현량을 측정한 결과, 기존의 BmA3 유전자 프로모터 대비 10% 가량 높은 발현 효율을 확인할 수 있었다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제32권7호
• /
• pp.922-929
• /
• 2019

Objective: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3'-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3'-UTR and pGL3 Control LDLR mutant-3'-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine premiR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3'-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR3'-UTR-driven (p<0.01), but not mutant LDLR-3'-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = -0.656, p<0.05). Conclusion: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3'-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.40-40
• /
• 2003

형질전환체 제작 시 발현벡터가 삽입되는 위치에 따라 발현 또는 억제되는 현상인 ‘position effect(위치효과)’를 극복하기 위해 Matrix Attachment Region(MAR)을 포함하는 발현벡터를 제작하였다 MAR는 핵 기질(nuclear matrix) 부착 부위로 발현조절에 관여하는 전사인자 등이 존재하는 핵 기질 부착부위로, 삽입된 발현벡터가 전사활성을 할 수 있는 게놈 환경을 제공해 주어 형질전환 유전자 발현을 향상시켜 준다고 보고되고 있다. 본 연구에서는 사람에서 이미 분리되어 염기서열이 밝혀진 MAR를 PCR로 증폭하였다. 증폭된 1,270 bp의 human alpha-1-antitrypsin MAR와 1,080 bp human corticosteroid binding globulin promoter MAR를 T vector에 클로닝하여 염기서열을 확인했으며 발현벡터 클로닝에 사용하였다. 유용 유전자와 세포 형질전환에 사용할 선별 유전자로 neo를 포함하며, 그 외 벡터골격은 pGL3 control vector를 사용하여 기본 발현벡터를 제작하였다. 이 벡터에 MAR를 5', 3' 양쪽 또는 한쪽만 포함하도록 클로닝하였다. 이는 MAR의 위치에 따른 게놈내 삽입 및 발현효과를 확인하여 형질전환동물 생산용 발현벡터로 활용하고자 한다.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
• /
• pp.141-141
• /
• 1998

In order to study the effect of nitric oxide on the regulation of mouse cyplal expression, 5' flanking DNA of mouse cytochrome P450 lal was cloned into pGL3 basic vector encoding luciferase gene. pcyplal-Luc was transfected into Hepa I cells and various chemicals were treated. Luciferase activity was stimulated 1000 folds over that of control by TCDD (2,3,7,8-tetrachloro-p-dioxin) treatment and this stimulation was dose dependent. When SNP (sodium nitroprusside) which donates nitric oxide was administrated, this stimulatory effect of TCDD on luciferase activity was decreased. And LPS (lipopolysaccharide) which is an iNOS (inducible nitric oxide synthase) inducer also decreased the stimulatory effect of TCDD on luciferase. And iNOS inhibitor N$\^$G/-nitro-ι-arginine + TCDD treatment increased the stimulation effect of TCDD and this effect was abolished when ι-arginine was added to N$\^$G/-nitro-ι-arginine + TCDD treatment. When N$\^$G/-nitro-ι-arginine was concomitantly administrated with SNP or LPS to confirm the effect of nitric oxide, the inhibitory effect of SNP or LPS was abolished. These data strongly suggest that nitric oxide might be an inhibitory regulator on the cytochrome P450 lal gene expression in Hepa cells.

• Journal of Microbiology and Biotechnology
• /
• 제27권6호
• /
• pp.1098-1105
• /
• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

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

• Preventive Nutrition and Food Science
• /
• 제21권1호
• /
• pp.62-67
• /
• 2016

Green tea (Camellia sinensis) is one of the most popular beverages in the world and has been acknowledged for centuries as having significant health benefits. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and it has been reported to have health benefit effects. Peroxisome proliferator-activated receptor ${\gamma}$ coactivator $(PGC)-1{\alpha}$ is a crucial regulator of mitochondrial biogenesis and hepatic gluconeogenesis. The objective of this study was to investigate whether EGCG from green tea can affect the ability of transcriptional regulation on $PGC-1{\alpha}$ mRNA expression in HepG2 cells and 3T3-L1 adipocytes. To study the molecular mechanism that allows EGCG to control $PGC-1{\alpha}$ expression, the promoter activity levels of $PGC-1{\alpha}$ were examined. The $PGC-1{\alpha}$ mRNA level was measured using quantitative real-time PCR. The -970/+412 bp of $PGC-1{\alpha}$ promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. EGCG was found to up-regulate the $PGC-1{\alpha}$ mRNA levels significantly with $10{\mu}mol/L$ of EGCG in HepG2 cells and differentiated 3T3-L1 adipocytes. $PGC-1{\alpha}$ promoter activity was also increased by treatment with $10{\mu}mol/L$ of EGCG in both cells. These results suggest that EGCG may induce $PGC-1{\alpha}$ gene expression, potentially through promoter activation.

• 대한의생명과학회지
• /
• 제13권3호
• /
• pp.239-244
• /
• 2007

Hoxc8 is one of the homeotic developmental control genes regulating the expression of many downstream target genes, through which animal body pattern is established during embryonic development. In previous proteomics analysis, proliferating cell nuclear antigen (PCNA) which is also known as cyclin, has been implied to be regulated by Hoxc8 in F9 murine embryonic teratocarcinoma cell. When the 5' upstream region of PCNA was analyzed, it turned out to contain 20 Hox core binding sites (ATTA) in about 1.17 kbp (kilo base pairs) region ($-520{\sim}-1690$). In order to test whether this region is responsible for Hoxc8 regulation, the upstream 2.3 kbp fragment of PCNA was amplified through PCR and then cloned into the pGL3 basic vector containing a luciferase gene as a reporter. When the luciferase activity was measured in the presence of effector plasmid (pcDNA : c8) expressing murine Hoxc8, the PCNA promoter driven reporter activity was reduced. To confirm whether this reduction is due to the Hoxc8 protein, the siRNA against Hoxc8 (5'-GUA UCA GAC CUU GGA ACU A-3' and 5'-UAG UUC CAA GGU CUG AUA C-3') was prepared. Interestingly enough, siRNA treatment up regulated the luciferase activity which was down regulated by Hoxc8, indicating that Hoxc8 indeed regulates the expression of PCNA, in particular, down regulation in NIN3T3 cells. These results altogether indicate that Hoxc8 might orchestrate the pattern formation by regulating PCNA which is one of the important proteins involved in several processes such as DNA replication and methylation, chromatin remodeling, cell cycle regulation, differentiation, as well as programmed cell death.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권8호
• /
• pp.3767-3772
• /
• 2014

Introduction: MicroRNAs have emerged as post-transcriptional regulators that are critically involved in tumorigenesis. This study was designed to explore the effect of miRNA 133b on the proliferation and expression of TBPL1 in colon cancer cells. Methods: Human colon cancer SW-620 cells and human colon adenocarcinoma HT-29 cells were cultured. MiRNA 133b mimcs, miRNA 133b inhibitors, siRNA for TBPL1 and scrambled control were synthesized and transfected into cells. MiR-133b levels in cells and CRC tumor tissue was measured by real-time PCR. TBPL1 mRNA was detected by RT-PCR. Cell proliferation was studied with MTT assay. Western blotting was applied to detect TBPL1 protein levels. Luciferase assays were conducted using a pGL3-promoter vector cloned with full length of 3'UTR of human TBPL1 or 3'UTR with mutant sequence of miR-133b target site in order to confirm if the putative binding site is responsible for the negative regulation of TBPL1 by miR-133b. Results: Real time PCR results showed that miRNA 133b was lower in CRC tissue than that in adjacent tissue. After miR-133b transfection, its level was elevated till 48h, accompanied by lower proliferation in both SW-620 and HT-29 cells. According to that listed in http://www.targetscan.org, the 3'-UTR of TBPL1 mRNA (NM_004865) contains one putative binding site of miR-133b. This site was confirmed to be responsible for the negative regulation by miR-133b with luciferase assay. Further, Western blotting and immunohistochemistry both indicated a higher TBPL1 protein expression level in CRC tissue. Finally, a siRNA for TBPL1 transfection obviously slowed down the cell proliferation in both SW-620 and HT-29 cells. Conclusion: MiR-133b might act as a tumor suppressor and negatively regulate TBPL1 in CRC.

• 한국환경성돌연변이발암원학회지
• /
• 제24권3호
• /
• pp.121-127
• /
• 2004

Cytochrome P4501B1(CYP1B1) is known to be inducible by xenobiotic compounds such as policyclic aromatic hydrocarbon(PAH) and dioxins such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). And these induction of CYP1B1 is also regulated by many categories of chemicals. In order to investigate the effects of several chemicals on CYP1B1 gene expression in Hepa-I and MCF-7 cells, 5' flanking DNA of human CYP1B1 was cloned into pGL3 basic vector containing luciferase gene, and then transfected into these cells. After treatment of chemicals, the luciferase activity was measured. CYP1B1 enzyme metabolize PAHs and estradiol. CYP1B1 metabolize estradiol to 4-hydrozyestradiol that is considered as carcinogenic metabolite. Luciferase activity was induced about 20 folds over that control by 1 nM TCDD (2,3,7,8-tetrachloto-p-dioxin). Recent industrialized society, human has been widely been exposed to widespread environmental contaminants such as PAHs(polycyclic aromatic hydrocarbon) that are originated from the imcomplete combustion of hydrocarbons. PAHs are known to be ligands of the AhR(aryl hydrocarbon receptor). Induction of cytochrome P4501B1(CYP1B1) in cell culture is widely used as a biomarker for PAHs. Therefore we have studied the effect of PAHs in the human breast cancer cells MCF-7 to evaluate bioactivity of PAHs. We have used the United State of America EPA selected 13 different PAHs, PAHs mixtures and extracts from environmental samples to evaluate the bioassay system. We examined effects of PAHs on the CYP1B1-luciferase reporter gene and CYP1B1 mRNA level. Benzo(k)fluoranthene and dibenzo(a, h)anthracene showed strong response to CYP1B1 promoter activity stimulation, and also CYP1B1 mRNAs increase in MCF-7 cells in a concentration-dependent manner. Acenaphthene, anthracene, benzo(b)fluoranthene, fluorene, fluoranthene, anphthanlene, pyrene, phenanthrene and carbazole were weak responders in MCF-7 cells. RT-PCR analysis indicated that PAHs significantly up-regulate the level of CYP1B1 mRNA.