• Journal of Life Science
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• v.13 no.4
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• pp.541-550
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• 2003

E2 glycoprotein of hepatitis C virus (HCV) comprises a surface of viral particle together with E1 glycoprotein, and is thought to be involved in the attachment of HCV viral particle to receptor (s) on the permissible cells including hepatocytes, B cells, T cells, and monocytes. We constructed a phage library expressing cellular proteins of hepatocytes on the phage surface, which turned out to be 8.8${\times}$$10^5$ cfu of diversity and carried inserts in 95% of library. We screened both cDNA phage library and 12-mer peptide library to identify the cellular proteins binding to E2 protein. Some intracellular proteins including tensin and membrane band 4.1 which are involved in signal transduction of survival and cytoskeleton organization, were selected from cDNA phage library through several rounds of panning and screening. On the contrary, membrane proteins such as CCR7, CKR-L2, and insulin-like growth factor-1 receptor were identified through screening of peptide library. Phages expressing peptides corresponding to those membrane proteins were bound to E2 protein specifically as determined by neutralization of binding assay. Since it is well known that HCV can infect T cells as well as hepatocytes, we examined to see if E2 protein can bind to CCR7, a member of C-protein coupled receptor family expressed on T cells, using CCR7 transfected tells. Human CCR7 cDNA was cloned into pcDNA3.1(-) vector and transfected into human embryonic kidney cell, 293T, and expressed on the surface of the cell as shown by flow cytometer. Binding assay of E2 protein using CCR7 transfected cells indicated that E2 protein bound to CCR7 by dose-dependent mode, giving rise to the possibility that CCR7 might be a putative cellular receptor for HCV.

• Bulletin of the Korean Chemical Society
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• v.28 no.8
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• pp.1317-1321
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• 2007

Arginine conjugated PAMAM dendrimer, PAMAM-R was modified with propylene oxide via hydroxylation of primary amines of arginine residues. About 49 amines were detected to be converted to amino alcohols by 1H NMR. The newly synthesized polymer, PAMAM-R-PO was able to completely retard pDNA from a charge ratio of 2. The average diameter of PAMAM-R-PO polyplex was found to be 242 nm at a charge ratio of 30. The Zeta-potential value of PAMAM-R-PO polyplex was able to reach 20-30 mV over a charge ratio of 10. PAMAM-R-PO indicated higher cell viability than unmodified PAMAM-R on HeLa and 293 cells because of its hydroxylated amines. Transfection experiments on 293 cells showed that the transfection efficiency of PAMAM-R-PO was found to be 1.5-1.9 times higher than that of PEI25kDa at a charge ratio of 30. The polymer eventually displayed about 2 times greater transfection efficiency than PAMAM-R at the same charge ratio in the absence of serum. Therefore, we concluded that the modification of primary amines of PAMAMR to amino alcohols gives positive effects such as reduced cytotoxicity and enhanced transfection efficiency on 293 cells for gene delivery potency of PAMAM-R.

• KSBB Journal
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• v.19 no.5
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• pp.341-347
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• 2004

Several gene delivery therapies are being developed for treatment of serum protein deficiency. EPO is one of the most promising therapeutic agent for this treatment which is currently being investigated in depth. This study has the ultimate purpose of improving the gene delivery system for an increase of red blood cell production. A plasmid DNA was constructed smaller than other plasmids for an increase in penetration into animal cells, and two genes were cloned into each vector as a co-delivery system to express erythropoietin, and interluekin-3 or thrombopoietin, which can act on erythroid cell, thus activating hematopoiesis synergically. This co-delivery system has an advantage of decreasing the labour required for industrial production of DNA vaccine. A new plasmid vector, pVAC, in size 2.9 kb, was constructed with the essential parts from PUC 19 and pSectagB, which is much smaller than other plasmid vector and is the size of 2.9 kb. Co-delivery system was constituted by cloning human erythropoietin with each of human interluekin-3 gene or human thrombopoietin gene into both pVAC and pSectagB. As a result, the transfection efficiency of pVAC was higer than that of pSectagB in vitro, and hematocrit level of the mice injected with pVAC is higher than that of other mice. And co-delivery system, made of several plasmid DNAs, was expressed in vitro.

• Entomological Research
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• v.48 no.5
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• pp.384-389
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• 2018

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site-specific recombination of bacteriophage ${\lambda}$. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of $8.4{\times}10^6$ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus-induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi-induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.

• Journal of Periodontal and Implant Science
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• v.37 no.4
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• pp.859-869
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• 2007

Naked DNA and standard vectors have been previously used for gene delivery. Among these, PEI can efficiently condense DNA and has high intrinsic endosomal activities. The aim of this study is to investigate whether the cationic polycation PEI could increase the transfection efficiency of BMP expressing DNA using a vector-loaded collagen sponge model. BMP-2/pcDNA3.1 plasmid was constructed by subcloning human BMP-2 cDNA into the pcDNA3.1 plasmid vector. PEI/DNA complexes were prepared by mixing PEI and BMP-2/pcDNA3.1 and the constructed complexes were loaded into the collagen sponges. In vitro studies, BMSCs were transfected with the PEI/BMP-2/pcDNA3.1 complexes from collgen sponge. The level of secreted BMP-2 and alkaline phosphatase activities of transfected BMSCs were significantly higher in PEI/BMP-2/pcDNA3.1 group than in BMP-2/pcDNA3.1 group (p<0.05). Transfected BMSCs were cultured and mineralization was observed only in cells treated with PEI/BMP-2/pcDNA3.1 complexes. In vivo studies, PEI/BMP-2/pcDNA3.1/collagen, BMP-2/pcDNA3.1/collagen and blank collagen were grafted in skeletal muscle of nude mice. Ectopic bone formation was shown in PEI/BMP-2/pcDNA3.1/collagen grafted mouse 4 weeks postimplantation, while not in BMP-2/pcDNA3.1 grafted tissue. This study suggests that PEI-condensed DNA encoding for BMP-2 is capable of inducing bone formation in ectopic site and might increase the transfection rate of BMP-2/pcDNA3.1. As a non-viral vector, PEI offers the potential in gene therapy for bone engineering.

• Radiation Oncology Journal
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• v.19 no.4
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• pp.381-388
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• 2001

Purpose : This study was conducted to assess the effects of x-irradiation on the expression of the novel glutathione S-transferase K1 gene. Materials and methods : Human glutathione S-transferase K1 (hGSTK1) DNA was purified and ligated to a pcDNA3.1/Myc-His(+) vector for the overexpression of hGSTK1 gene. MCF-7 cells were transfected with or without the recombinant hGSTK1 gene, and irradiated with 6 MV x-ray. After incubation of 14 days, cell survival was measured and compared. The expression of hGSTK1 and the effect of x-irradiation on hGSTK1 expression were also estimated in MCF-7 cells transfected with or without the hGSTK1 gene by RT-PCR. Results : Following 2 to 12 Gy of x-irradiation, the cell survivals were higher in the MCF-7 cells transfected with the hGSTK1 gene than in those without transfection. Despite the higher cell survival in the hGSTK1-transfected cells, RT-PCR for hGSTK1 mRNA revealed no significant differences according to radiation dose, fractionation, and time after irradiation. Conclusion : The MCF-7 cells transfected with the hGSTK1 gene showed higher cell survival than those without transfection of the gene. The hGSTK1 gene might be associated with the radiosensitivity of MCF-7 cell line and further analysis should be needed.

• Bulletin of the Korean Chemical Society
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• v.25 no.7
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• pp.1025-1030
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• 2004

A hybrid linear polymer-dendrimer block copolymer, poly(ethylene glycol)-block-poly(L-lysine) dendrimer, was synthesized and introduced to form polyionic complexes with DNA. The copolymer formed core-shell type nanoparticles with plasmid DNA. From dynamic light scattering experiments, the mean diameter of the polyplexes was observed to be 154.4 nm. The complex showed much increased water solubility compared to poly(L-lysine). The plasmid DNA in polyplexes was efficiently protected from the enzymatic digestion of DNase I. The cytotoxicity and transfection efficiency for 293 cells was measured in comparison with poly(Llysine).

• YAKHAK HOEJI
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• v.46 no.2
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• pp.143-148
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• 2002

The human polyomavirus JC virus is the etiologic agent of progressive multifocal leukoencephalopathy (PML). The JC virus early promoter directs cell-specific expression of the viral replication factor large T antigen, thus transcriptional regulation constitutes a major mechanism of glial tropism in PML. Here we found that pentanucleotide sequence immediately upstream of the TATA sequence functions as a cell-specific silencer in the JC virus transcription. In vitro binding studies showed that synthetic oligonucleotides spanning a pentanucleotide sequence, designated "oligo 2", interacts with nuclear proteins from non-glial cells in a cell-specific manner. Furthermore, the sequence preferentially repressed the heterologous thymidine kinase promoter activity in non-glial cells. We also tested whether JC virus transcription is controlled by DNA methylation. Transient transfection of in vitro methylated JC virus promoter abolished transcription in both the glial and non-glial cells. The repression fold was much larger in glial cells than in non-glial cells. Taken together, this finding suggests that glial cell-specific expression of the JC virus is controlled by DNA methylation as well as cell-specific silencers.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.588-591
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• 2000

Serine palmitoyl transferase(SPT) and ceramidase are the key enzymes in sphingolipid biosynthesis. To study sphingolipid metabolism, we have got the 5'-upstream regions of human serine palmitoyl transferase subunit II and acid ceramidase gene by using GenomeWalker kits(Clontech Co.). Human genomic DNA was purified from HT29, human colon canser cell line by using DNAzol. We got several bands after secondary PCR and subcloned them to T7bule vector. Human SPTII promoter which we got was 2690bp but we cut it with Bgl II and vector with Bgl II and BamH I, and subcloned 1782bp to pGL2-enhancer vector and pGL2-basic vector with luciferase reporter gene. Human acid ceramidase promoter which we got were 2028bp and 1034bp and subcloned to pGL2-enhancer vector and pGL2-basic vector. We transfected these promoters to HT29 cell and assayed luciferase activity. For measuring transfection efficiency, pRL-TK vector with seapancy luciferase reproter gene was cotransfected with these promoters.

• Biomedical Science Letters
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• v.6 no.1
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• pp.73-82
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• 2000

Blunt-end DNA fragments can be inserted in two different orientations. Conventionally, their directions are determined by restriction enzyme digestion or by DNA sequencing, however, these methods are often limited in their use due to the lack of appropriate enzyme sites or large sample numbers, respectively. In the present study, a novel strategy and the corresponding protocol for the simple determination of insert orientation is introduced. Using conventional sequencing primers and PCR primers that have been used for amplification of the insert, single clones, which have inserted the fragment in the desired orientation, were easily identified by this PCR-based method. The fidelity of this system was confirmed by cloning of a tar urocortin cDNA, which is a recently discovered neuropeptide. Recombinant clones identified by this method were further shown to be fully functional, and using these, for the first time, urocortin was recombinantly expressed in eukaryotic cells.