• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• 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.

• Journal of Aquaculture
• /
• v.11 no.4
• /
• pp.457-463
• /
• 1998

An expression vector, pUC19N6-luc, containing nuclear matrix attachment region(MAR) isolated from Misgurnus mizolepis liver and control expressino vector, pUC19-luc, were constructed. After these vectors were transferred into CHSE-214 cell line by electroporation, the expression rate of luckferase gens, copy number of vectors and chromosome integration of vectors were analyzed by using assay of luciferase activity, PCR and Southern blotting. While the expression pattern of luciferase gene of pUC19-luc was shown in typicla transient ecpression pattern, that of pUC19N6-luc was highly increased at the 5 days after transfectrion. Although the cope number of pUC19N6-luc vector was higher than that of pUC19-luc vector, these vectors were integrated into chromosome at the same time point in the transfected CHSE-214 cells. In conclusion, the increase of luciferase gene expression of pUC19N6-luc was resulted from not the maintaining of the high copy number but the formation of transcription-favorable structure by MAR effect after chromosomal integration.

• Microbiology and Biotechnology Letters
• /
• v.35 no.3
• /
• pp.184-190
• /
• 2007

Approximately 2.0 kb of the promoter region of the Pichia pastoris phosphoglycerate kinase gene (PGK1) was reduced to a 266 bp fragment and this minimized portion was used for construction of a new episomal constitutive expression vector in P. pastoris. As an approach to developing a constitutive expression vector in P. pastoris, the GAP promoter region of the Pichia expression vector pGAPZB was replaced with sequentially deleted PGK1 promoter fragments fused to a beta-galactosidase gene. When a lacZ gene was used as a reporter gene, PGK1 promoter strength was lower than that of the constitutive GAP promoter but it was higher than TEF1. We report here the development of the pPGKZ-E vector as a new episomal expression vector for heterologous gene expression by removing non-essential regions of the PGK1 promoter. This broadens the choice of episomal expression vectors for controlled constitutive expression in P. pastoris.

• Journal of Life Science
• /
• v.20 no.9
• /
• pp.1314-1323
• /
• 2010

In this study, we constructed various kinds of binary vectors with the pPZP backbone for co-overexpression, tissue- or development-specific expression and stress-inducible expression, and validated them for ectopic expression of target genes. Using a modified CaMV 35S promoter, a binary vector was generated for co-overexpression of two different genes and was confirmed to be efficient for overexpressing two different target genes at the same time and place. Binary vectors containing At2S3, KNAT1 or LFY promoters were constructed for tissue-specific or development-specific gene expression, and the binary vectors were suited for embryo/young seedling stage-, shoot apical meristem- or leaf primordia-specific expressions. Furthermore, the binary vectors containing RD29A or AtNCED3 promoters were validated as suitable vectors for gene expression induced by abiotic stresses such as high salt, ABA, MV and low temperature. Taken together, the binary vectors constructed in this study would be very useful for analyzing the biological functions of target genes and molecular mechanisms through ectopic expression.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
• /
• 2018.10a
• /
• pp.444-447
• /
• 2018

Baculovirus expression systems have many known advantages including fast and cost-effective methods to generate large amounts of recombinant proteins in comparison to bacterial expression systems, particularly those requiring complex post-translational modifications. Especially, recombinant baculoviruses can transfer their vectors and express their recombinant proteins in a wide range of mammalian cell types. In this study, baculoviral vectors which were reconstructed from pcDNA3.1 vector, were recombined with cytomegalovirus (CMV) promoter,uroplakin II promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), and protein transduction domain (PTD). These recombinant vectors were infected with various cells and cell lines. The baculovirus vector thus developed was analyzed by comparing the metastasis and expression of the recombinant genes with conventional vectors. These results suggest that the baculovirus vector has higher efficiency in metastasis and expression than the control vector.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
• /
• 2016.10a
• /
• pp.923-926
• /
• 2016

Baculovirus vectors were reconstructed using cytomegalovirus (CMV) promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), and protein transduction domain (PTD) genes. These reconstructed vector was transfected into various cell lines and tissues. We compared this reconstructed vector with other control vectors in view of gene transfer and gene expression. In conclusion, we confirmed that gene transfer and expression of these reconstructed vectors was higher efficient than any other control vector.

• Microbiology and Biotechnology Letters
• /
• v.36 no.4
• /
• pp.260-267
• /
• 2008

The choice of expression vector is very important for industrial production of proteins. Therefore, the systematic mining of promoters over a wider range of genetic resource and/or host is required. We previously reported a novel bidirectional reporting system (pBGR) for the isolation of promoters from metagenome and screened useful promoters that functioned constitutively in E. coli under general culture conditions. Among them, three promoter sequences including each upstream region were amplified by PCR and used to construct new expression vectors. To facilitate subcloning, a multi-cloning site was incorporated into the downstream region of the revere primer sequence. At these sites, GFP, esterase and $\beta$-glucosidase were subcloned and analyzed the constitutive expression ability of new promoter in terms of protein solubility and expression level. As a result, these vectors expressed the proteins constitutively to a level of $2{\sim}3%$ of the total cell protein in soluble fraction (>80 %). This study suggested that excavation of metagenomic promoters for construction of expression vector in a certain strain could provide a way for the development of the expression systems.

• Journal of Life Science
• /
• v.19 no.6
• /
• pp.809-817
• /
• 2009

The phenotypes associated with a gene function are often the best clue to its role in the plant. Transgenic plants ectopically expressing or suppressing a gene can provide useful information related to the gene function. In this study, we constructed three vectors - pFGL571, pFGL846 and pFGL847 - for the Agrobacterium-mediated ectopic expression of plant genes using pPZP211 and modified CaMV 35S, UBQ3 or UBQ10 promoters. The three vectors have several merits such as small size, high copy in bacteria, enough restriction enzyme sites in multi cloning sites and nucleotide sequence information. Analysis of transgenic plants containing GUS or sGFP reporter genes under the control of modified CaMV 35S, UBQ3 or UBQI0 promoter revealed that all of the three promoters showed high activities during most developmental stages after germination and in floral organs. Furthermore, we generated a RNAi module vector, pFGL727, to suppress plant gene expressions and confirmed that pFGL727 is useful for the suppression of a gene expression using rice transgenic plants. Taken together, our new vectors would be very useful for the ectopic expression or the suppression of plant genes.

• Applied Biological Chemistry
• /
• v.46 no.1
• /
• pp.7-11
• /
• 2003

Cloning and expression of copper-binding peptide gene in E. coli was carried out to enhance the copper-chelation capacity. E. coli was transformed with pET vector containing the copper-binding region of potato polyphenol oxidase gene and polyhistidine-coding DNA, and the copper content of E. coli harboring each vector was measured. No increase in intracellular copper was observed in E. coli harboring PPOCBpET32 vector, which contains DNA for polyphenol oxidase copper-binding region. Intracellular copper content of E. coli harboring pE728a vector, which contains one hexahistidine unit DNA, was 2,500 ppm after culturing without kanamycin, whereas E. coli harboring pET-his vector, which contains nine hexahistidine unit DNAs was 3,200 ppm.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
• /
• 2018.05a
• /
• pp.588-591
• /
• 2018

Baculovirus was originally isolated from the alfalfa looper and contains a 134-kbp genome with 154 open reading frames (ORF). The major capsid protein VP39 together with some minor proteins forms the nucleocapsid ($21nm{\times}260nm$) that encloses the DNA with p6.9 protein. They are double-stranded, circular, supercoiled DNA molecules in a rod-shaped capsid. Wild-type baculoviruses exhibit both lytic and occluded life cycles that develop independently throughout the three phases of virus replication. Recombinant baculoviruses can transfer their vectors and express their recombinant proteins in a wide range of mammalian cell types. Especially, inclusion of a dominant selectable marker in these baculoviral vectors can express diverse recombinant genes in many cells. Baculoviral vectors were reconstructed with cytomegalovirus (CMV) promoter,uroplakin II promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), protein transduction domain (PTD) gene and so on. These reconstructed vectors were infected into various cell and cell lines. We performed transfection and expression of these recombinant vectors comparison with other control vectors. From this study, we knew that transfection and expression of these recombinant vectors have higher efficacy than any control vector. This work was supported by a grant from Mid-Career Researcher Program(NRF-2016R1A2B4016552) through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(MSIP).