• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.2
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• pp.145-149
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• 2004

The expression of a fusion protein comprised of the B. thuringiensis crystal protein, Cry1Ac, and enhanced green fluorescent protein (EGFP) in Escherichia coli XLl-blue was examined. Three recombinant plasmids were transformed into E. coli XL1-blue and named as ProAc/Ec, MuEGFP/Ec and ProMu-EGFP/Ec, respectively. All transformants were observed by light and fluorescence microscopy at mid-log phase. The expression in E. coli transformants, ProMu-EGFP/Ec and MuEGFP/Ec, exhibited bright enough fluorescence to be observed. Furthermore, ProMu-EGFP/Ec produced fluorescent inclusions, which may have been recombinant crystals between EGFP and Cry1Ac while MuEGFP/Ec expressed soluble EGFP in cell. In SDS-PAGE, ProAc/Ec had 130 kDa crystal protein band and MuEGFP/Ec had thick 27 kDa EGFP band. However, ProMu-EGFP/Ec had about 150 kDa fusion protein band. Accordingly, these results indicated that a fusion protein between the B. thuringiensis crystal protein and a foreign protein under the lacZ promoter was successfully expressed as granular structure in E. coli. It is suggested that the E. coli expression system by N-terminal fusion of B. thuringiensis crystal protein may be useful as excellent means for fusion expression and characterization of B. thuringiensis fusion crystal protein.

• Journal of Veterinary Science
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• v.22 no.6
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• pp.76.1-76.15
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• 2021

Background: The development of a vaccine for Jembrana disease is needed to prevent losses in Indonesia's Bali cattle industry. A DNA vaccine model (pEGFP-C1-tat) that requires a functional delivery system will be developed. Polylactic-co-glycolic acid (PLGA) may have potential as a delivery system for the vaccine model. Objectives: This study aims to evaluate the in vitro potential of PLGA as a delivery system for pEGFP-C1-tat. Methods: Consensus and codon optimization for the tat gene was completed using a bioinformatic method, and the product was inserted into a pEGFP-C1 vector. Cloning of the pEGFP-C1-tat was successfully performed, and polymerase chain reaction (PCR) and restriction analysis confirmed DNA isolation. PLGA-pEGFP-C1-tat solutions were prepared for encapsulated formulation testing, physicochemical characterization, stability testing with DNase I, and cytotoxicity testing. The PLGA-pEGFP-C1-tat solutions were transfected in HeLa cells, and gene expression was observed by fluorescent microscopy and real-time PCR. Results: The successful acquisition of transformant bacteria was confirmed by PCR. The PLGA:DNA:polyvinyl alcohol ratio formulation with optimal encapsulation was 4%:0.5%:2%, physicochemical characterization of PLGA revealed a polydispersity index value of 0.246, a particle size of 925 nm, and a zeta potential value of -2.31 mV. PLGA succeeded in protecting pEGFP-C1-tat from enzymatic degradation, and the percentage viability from the cytotoxicity test of PLGA-pEGFP-C1-tat was 98.03%. The PLGA-pEGFP-C1-tat demonstrated luminescence of the EGFP-tat fusion protein and mRNA transcription was detected. Conclusions: PLGA has good potential as a delivery system for pEGFP-C1-tat.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.205-214
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• 2002

There are many methods to introduce exogenous DNA into embryo to produce transgenic animals. Exogenous gene can be integrated into oocyte by sperm vector. In this study, sperm was used as a vector for a transgene, which is encoding enhanced green fluorescent protein (EGFP). The objective of this study was to investigate the expression of exogenous gene in bovine embryos after injection of spermatozoa cocultured with EGFP DNA fragment. Spermatozoa were plunged into liquid nitrogen and thawed several times or shook in 0.2% Triton X-100 to remove sperm membrane followed by DTT treatment. The injected oocytes were co-cultured with vero cells in CR1aa, and expression of EGFP gene was observed under fluorescent microscope. Blastocyst formation rates of oocytes injected with sperm treated with DTT, DTT-freezing or DTT-Triton X-100 were 34.7, 39.4 and 31.9%, respectively. The rates of EGFP expression in oocytes injected with 54 ng DNA after DTT-treated, DTT-freezing and DTT-Triton X-100-treated sperm were 0, 19.1 and 13.9%. On the other hands, expression rate of oocytes injected with sperm cocultured with 13.5, 27 and 63.5 ng of EFGP DNA were 6.7, 9.0 and 5.1%, respectively. When intact sperm was mixed with 63.5 ng/${mu}ell$ EGFP DNA fragment, and then electroporated before injection, the expression rate of injected oocyte was 2%. Unexpectedly, electro-poration could not increase the expression rate. These results suggest that sperm can be used as a transgene vector, even if the efficiency was low (19.1%).

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.305-315
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• 2001

In this study, the production of transgenic embryo was attempted by microinjection or round spermatid cultured with foreign DNA. At first, the expression of haploid spermatids specific gene, mTP1 in mouse and hPrm2 in hamster spermatids were investigated by RT-PCR method in testes of young mice and hamster testis. The specific gene expression first appeared at 18 days post partum (dpp) in mice spermatid and 20 dpp in hamster spermatid. Therefore, the round spermatids isolated from 17 dpp mice and 19 dpp hamster were used for the introduction of foreign EGFP gene into haploid round spermatids. For the introduction of EGFP gene haploid round spermatids suspended in medium including EGFP gene were treated with a different electric field strength at 0.11, 0.18 and 0.44 ㎸/cm. After electrical stimulation, viability of testicular sperm cells and 67.6%, 66.4% and 49.9%, in mice and 62.6%, 57.9% and 27% in hamster, respectively. These values were significantly lower than those of non-treated control groups 80.5% in mouse and 69.1% in hamster After 72 hrs culture, the highest expression rate of EGFP gene, 28.5% in mice and 32.1% in hamster were obtained from tile spermatogenic cells electroporated by the field strength or 0.18 ㎸/cm. Then, the ability of fertilization and embryonic development of haploid spermatids transfected with foreign EGFP gene were estimated by the microinjection of spermatids into hamster oocytes. The Irate pronuclear formation rate (77.5%) was lower than non-treated control (80%), and the cleavage rate of the treated group (58.8%) was lower than control (65%). To prove the foreign EGFP integration in hamster embryos, 2-cell stage hamster embryos were subjected to the observation under the fluorescence microscope, and the PCR analysis. As a result, about 44% of 2-cell embryos were showed the integration of EFGP gene into their genome. Therefore, These results suggest the possibility to produce transgenic hamsters by microinjection of haploid spermatid transfected with foreign DNA.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.115-118
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• 2000

Recombinant plasmids harboring a heterologous gene coding for the enhanced green fluorescent protein (EGFP) were transfected and expressed in Drosophila melanogaster S2 cells. A stable transformation of polyclonal cell populations expressing EGFP were isolated after 4 weeks of selection with hygromycin B. The recombinant EFGP expressed in transformed S2 cells consisted of a molecular weight of 27 kDa. EGFP expression was also confirmed by fluorometric measurement. The maximum EGFP concentration was about 9.3 mg/I. The present findings demonstrate not only the successful stable expression of EGFP in Drosophuila was about 9.3 mgI. The present findings demonstrate not only the successful stable expression of EGFP in Drosophila S2 cells, but also the use of EGFP as a reporter to analyze gene expression, with its potential of a Drosophila cell expression system for recombinant protein production being an alternative to a baculovirus-insect cell expression system.

• Neonatal Medicine
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• v.15 no.1
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• pp.22-31
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• 2008

Purpose : Bronchopulmonary dysplasia (BPD) is characterized by arrested vascular and alveolar growth in the premature lung. Considering the consequences of arrested lung growth, the idea of administering bone marrow cells to enhance the inborn repair mechanism is promising as this may reduce the morbidity and mortality of BPD. We followed enhanced green fluorescent protein (EGFP)-labeled bone marrow cells (BMC) injected intraperitoneally into non-EGFP mice in order to determine their fate after transplantation. Methods : An angiogenesis inhibitor, SU1498, was injected subcutaneously on day 3 in non-EGFP C57BL/6 newborn mice to create a model of arrested alveolar development. On the following day, $1{\times}10^6$ BMCs isolated from major histocompatibility complex (MHC)- matched syngenic EGFP mice were injected intraperitoneally to non-EGFP BPD mice. Morphometric analysis, immunostaining, and confocal microscopy were performed to determine the fate of EGFP-positive stem cells in the injured lung. Results : SU1498 injection reduced alveolar surface area and mean alveolar volume in newborn mice. BMC injection resulted in recovery of lung structure comparable to controls. EGFP-positive BMCs were identified in the lungs of the recipient mice after intraperitoneal injection. The injected EGFP cells were co-stained with endothelial and epithelial cells of the developing lung as determined by confocal microscopy. Conclusion : Our results illustrated that EGFP-positive BMCs engrafted and trans-differentiated into epithelial and endothelial cells after intraperitoneal injection in a mouse model of arrested alveolar development.

• Journal of Aquaculture
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• v.13 no.1
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• pp.29-37
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• 2000

In previously study we isolated several fish matrix attachment regions (MARs) capable of replicating the plasmid by itself. In this study we construct a fish expression vector pBaEGFP(+) containing mud loach ${\beta}$-actin promoter EGFP as reporter gene and SV40 signal. To analyze the effects of the fish expression vector respectively. The fish ARS containing constructs pBaEGFP(+)-ARSs were transfected cells with pBaEGFP(+)-ARS101 and pBaEGFP(+)-ARS223 reduced 10 days to 25 days and then was constant to 30 days after transfection while that of the control vector without ARS element was basal level. The intensity of both constructs showed about 30fold of the intensity compared with the control vector on 30days after transfection individually .E. coli back-transformation analysis shows that pBaEGFP(+)-ARS223 and pBaEGFP(+)-ARS905 maintain in episomal state at least 30 days after transfection. The result indicates that both may be able to replicate the vector in BF-2 cell. Therefore the matrix-attached ARSs enhancing expression of the reporter gene might be useful as a component o the expression vector for transgenic studies.

• Journal of Life Science
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• v.26 no.8
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• pp.875-880
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• 2016

The plasma membrane plays a crucial role in relaying signals from the outside environment to the inside of the cells. In eukaryotic cells, the inner leaflets of the plasma membrane are composed mostly of phospholipids, including phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositides (PIs). In this study, we tried to analyze the morphological changes induced by EGFP-fused membrane binding proteins, which are targeted to the plasma membrane via specific phospholipids binding. As a result, we found that overexpression of EGFP-P4M-SidM, a specific PI4P binding protein, or EGFP alone, did not induce any morphological changes. On the other hand, overexpression of EGFP-PLCδ1(PH), which is a specific PI(4,5)P₂ binding protein, EGFP-AKT1(PH) which binds to PI(3,4,5)P₃, or EGFP-OSH2(PH)×2 which binds to PI4P and PI(4,5)P₂, could induce the filopodia and lamilapodia formation as well as cell shrinkage. Overexpression of Lact-C2-EGFP which is a specific PS-binding probe, EGFP fused Aplysia phosphodiesterase 4 (ApPDE4) long-form (L(N20)-EGFP) which is localized to the plasma membrane via hydrophobic interaction, or EGFP fused Aplysia PDE4 short-form (S(N-UCR1-2)-EGFP) which is localized to the plasma membrane via electrostatic interaction, could induce cell shrinkage, but not filopodia or lamilapodia formation. Taken together, our data support that the different phospholipid bindings in the plasma membrane could induce different characteristic morphological changes. Thus, we can analyze, characterize, and classify the cellular morphological changes induced by the various phospholipid binding proteins.

• Nuclear Medicine and Molecular Imaging
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• v.41 no.3
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• pp.226-233
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• 2007

Purpose: Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. Materials and Methods: A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. Results: The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, $T_{1/2}$ of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. Conclusion: A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene.

• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.