• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2017.05a
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• pp.700-703
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• 2017

Baculovirus vectors were recombined using uroplakin II promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), protein transduction domain (PTD) gene and so on. These novel recombinant vectors were infected into various cell lines. We performed and analyzed gene transfer and gene expression of these recombinant vectors comparison with other control vectors. From this result, we identified that these recombinant vectors have higher efficient gene transfer and expression of than control vector.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2016.05a
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• pp.711-714
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• 2016

Recombinant baculovirus was reconstructed with useful genes of polyhedron promoter, vesicular stomatitis virus G (VSVG), polyA, cytomegalovirus (CMV) promoter, enhanced green fluorescent protein (EGFP), and protein transduction domain (PTD). This reconstructed vector was infected into various cell lines and tissues. We investigated gene transfer and gene expression of this reconstructed vector in comparison to other vectors and recognized that this reconstructed vector was higher effective than any other control vector.

• BMB Reports
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• v.40 no.4
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• pp.547-553
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• 2007

Many motifs along the 1.2 kb myostatin promoter (MSTNpro) in sheep have been found by the MatInspecter program in our recent study. To further verify the role of the motifs and better understand the transcriptional regulation mechanism of the myostatin gene in sheep, the reporter gene EGFP (enhanced green fluorescent protein) was selected and the wild-type (W) vector MSTNPro$^W$-EGFP or motif-mutational (M) vector MSTNPro$^M$-EGFP were constructed. The transcriptional regulation activities were analyzed by detecting the fluorescence strength of EGFP in C2C12 myoblasts transfected with the vectors. The results showed that E-box (E) 3, E4, E5 and E7, particularly E3, E5 and E7, had important effects on the activity of the 1.2 kb sheep myostatin promoter. In addition, we also detected several other important motifs such as MTBF (muscle-specific Mt binding factor), MEF2 (myocyte enhancer factor 2), GRE (glucocorticoid response elements) and PRE (progesterone response elements) along the sheep myostatin promoter by the mutational analysis.

• Journal of Microbiology
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• v.40 no.4
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• pp.295-300
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• 2002

Previously, we reported induced expression of developmentally regulated CTP-binding protein 2 (DRG2) in fish cells at the late stage of rhabdovirus infection. To investigate the biological role of fish DRG2 (fDRG2), we transfected CHSE-214 cells with an expression vector containing complete fDRG2 fused to the N-terminal end of an enhanced green fluorescent protein (EGFP). Low level expression of fDRG2-EGFP did not induce morphological change or cell death. However, a high level expression of fDRG2-EGFP induced cell rounding and caused depletion of the cell population in FACS analysis. Several truncated fragments were fused to EGFP. FACS analysis was conducted to determine the presence of cells expressing high levels of the resulting chimera. While cells expressing a high level of N-terminus were detected, those expressing high levels of the C-terminal fragment 243-290 containing the G4 motif were absent in FACS analysis. Based on these observations, we propose that overexpression of fDRG2 may induce cell rounding, a representative cytopathic effect of virus-infected cells in the late stage of infection and the C-terminus of the fDRG2 is essential for this function.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.339-347
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• 2001

Sperm-mediated DNA transfer has a potential to markedly simplify techniques for the generation of transgenic animals. The exogenous DNA transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently introduced in the production of transgenic animals. In this study, the developmental competence and tile expression rates of transgene were investigated after injection of spermatozoon or sperm head with enhanced green fluorescent protein (EGFP) gene into the mature porcine oocytes. The porcine oocytes were injected with intact sperm, membrane-disrupted sperm or sperm head. After injection. embryos were cultured in NCSU23 medium up to the blastocyst stage, and the developmental competence and expression rates were studied. The developmental rate (67.0%) of sperm injection group was higher than that (59.7%) of sperm head injection group, and the rates of EGFP expression were also significantly different between sperm injection and sperm head injection groups (42.1 vs 20.0%) (F＜0.05). In the porcine oocytes injected with sperm treated with different methods of membrane disruption, the removal of sperm membrane did not alter the developmental competence of embryos. The rate of blastocysts at 7 days after injection with intact and membrane disrupted sperm were 15.0 and 14.2%, respectively. The EGFP expression rates, 38.4% in embryos injected with frozen-thawed sperm was higher than that, 22.4% of embryos injected with the Triton X-100 treated sperm. Prior to injection, sperm were cultured in different EGFP gene concentrations from 0.Ol to 1ng/u${mu}ell$. However, no significant difference in developmental rates of embryos among different concentrations of EGFP gene were observed. The highest expression rate of EGFP gene, 37.4% was obtained from the embryos injected with spermatozoa treated with 0.1 ng/${mu}ell$ EGFP gene. These results suggested that exogenous DNA could be attached to the membrane disrupted sperm, and that these sperm could be used as a vector carrying foreign DNA into embryos.

• Journal of Life Science
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• v.18 no.7
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• pp.912-917
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• 2008

To investigate the biological activity of recombinant human granulocyte colony-stimulating factor (rec-hG-CSF) in mammalian cells, hG-CSF gene was cloned using the eDNA extracted from the human squamous carcinoma cell lines and rec-hG-CSF was produced in CHO cell lines. To analyze the biological activity in vivo, the rec-hG-CSF protein was injected into mice subcutaneously on days 0 and 2. Blood was withdrawn for white blood cell (WBC) determination 5 days after the first injection. WBC values were found to have increased significantly. A pEGFP-mUII-hG-CSF vector was transfected into somatic cell lines isolated from bovine fetal cells. The colony expressing EGFP signals was observed with a confocal microscope. These data suggest that the rec-hG-CSF produced in this study has potent activity in vivo. Thus, the results of this biological activity show that rec-hG-CSF can be enhanced considerably by genetic engineering that affects potential activity, including mutations, which add the oligosaccharide chain and construct double-fusion proteins. A pEGFP-mUII-hG-CSF vector can be utilized for the production of cloned transgenic livestock.

• Molecules and Cells
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• v.26 no.1
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• pp.87-92
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• 2008

We employed dual color Fluorescence Cross Correlation Spectroscopy (FCCS) to measure the interaction between PKA regulatory (RII) and catalytic subunits (CAT) in living cells. Elevation of intracellular cAMP with forskolin decreased the cross-correlation amplitude between RFP-fused RII (RII -mRFP) and GFP-fused CAT (CAT-EGFP) by 50%, indicating that cAMP elevation leads to dissociation of RII-CAT complexes. Moreover, diffusion coefficient analysis showed that the diffusion rate of CAT-EGFP was significantly increased, suggesting that the decreased RII-CAT association caused by cAMP generated free CAT subunits. Our study demonstrates that in vivo FCCS measurements and their quantitative analysis permit one not only to directly quantify protein-protein interactions but also to estimate changes in the intracellular cAMP concentration.

• BMB Reports
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• v.41 no.10
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• pp.716-721
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• 2008

To explore the effects of deregulated expression of the EBNA1 binding protein 2 (EBP2) on cell growth, we generated human HEK293 stable clones constitutively expressing an EBP2-EGFP fusion protein. We found both RNA and protein levels of cyclin E1, a dominant oncoprotein, were elevated in the EBP2- EGFP stable clones. These findings were confirmed by flow cytometry bivariate analysis of cyclin expression versus DNA content. Moreover, the increase in p21 expression and the specific phosphorylation at Ser1981 of ATM and Ser15 of p53 were also observed in these stable clones, and these observations may explain the failure to observe an increase in Cdk2 kinase activity. In addition, after one year of passage culture, the EBP2-EGFP stable clones tended to lose 4 to 5 chromosomes per cell when compared to that of control cells. All of these findings provide a possible link between deregulated expression of EBP2 and tumor development.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2013.05a
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• pp.1006-1008
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• 2013

A novel recombinant baculovirus vector system containing coding genes for polyhedron promoter, vesicular stomatitis virus G (VSVG), polyA, cytomegalovirus (CMV) promoter, enhanced green fluorescent protein (EGFP), and protein transduction domain (PTD) was constructed. We applied this recombinant baculovirus vector into cells and murine tissues and compared efficacy of gene transfer and expression of this recombinant baculovirus vector system with control vector system. From this result, we confirmed that this novel recombinant baculovirus vector system was very effective than control vector system.

• Journal of Life Science
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• v.28 no.2
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• pp.162-169
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• 2018

Many cytoplasmic proteins are targeted to the cytoplasmic membrane of the trans-Golgi network (TGN) via an N-terminal short helix. We previously showed that the 20 N-terminal amino acids of Aplysia phosphodiesterase 4 (ApPDE4) long form are sufficient for its targeting to the plasma membrane and the TGN. The N-terminus of the ApPDE4 long form binds to PI4P and sulfatide in vitro. Therefore, in order to decipher the roles of sulfatide in Golgi complex targeting, we examined the cellular localization of sulfatide-binding peptides. In this study, we found that enhanced green fluorescent protein (EGFP) fused to the C-terminus of modified sulfatide- and heparin-binding peptides (mHSBP-EGFP) was localized to the TGN. On the other hand, its mutant, in which tryptophan was replaced with an alanine, leading to the impairment of heparin and sulfatide binding, was localized to cytosol. We also found that the TGN targeting of mHSBP-EGFP is impaired by the treatment of antimycin A, phenylarsine oxide (PAO), and adenosine but not a high concentration of wortmannin. These results suggest that PAO and adenosine-sensitive kinases, including phosphatidylinositol 4-kinase II, may play key roles in the recruitment of mHSBP-EGFP.