• BMB Reports
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• 제44권1호
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• pp.40-45
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• 2011

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects cells and tissues from extracellular damage by eliminating superoxide anion radicals produced during metabolism. Two different forms of EC-SOD exist, and their different enzyme activities are a result of different disulfide bond patterns. Although only two folding variants have been discovered so far, five folding variants are theoretically possible. Therefore, we constructed five different mutant EC-SOD expression vectors by substituting cysteine residues with serine residues and evaluated their expression levels and enzyme activities. The mutant EC-SODs were expressed at lower levels than that of wild-type EC-SOD, and all of the mutants exhibited inhibited extracellular secretion, except for C195S ECSOD. Finally, we demonstrated that co-expression of wild-type EC-SOD and any one of the mutant EC-SODs resulted in reduced secretion of wild-type EC-SOD. We speculate that mutant EC-SOD causes malfunctions in systems such as antioxidant systems and sensitizes tissues to ROS-mediated diseases.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2007년도 International Meeting of the Microbiological Society of Korea
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• pp.83-85
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• 2007

Deep-sea bacteria are adapted to extreme environments, such as high pressures and cold temperatures. We have isolated many piezophiles which grow well even under high pressures from deep-sea sediment. Shewanella violacea DSS12 and Moritella japonica DSK1 have the ability to grow at up to 70 MPa, and those bacteria have unique mechanisms of gene expression in response to high pressure conditions. The combination of gene expression systems in piezophiles, like the high pressure-dependent promoters and GFP reporter gene, may reveal highly fluorescent cells when exposed to high hydrostatic pressure conditions. It is predicted that a novel bio-sensing system can be made to probe high pressure environments using living bacteria. First, gene transformation into our piezophiles, strains DSS12 and DSK1, were examined. Eschericha coli S17-1 was used for bacterial conjugation with those piezophiles. As a result, the broad host range vector, pKT231, and the shuttle vector, pTH10, were successfully introduced to DSS12 and DSK1, respectively. Next, The pressure regulated promoters from DSS12 and DSK1 were cloned into proper vectors and combined with GFP as a reporter gene downstream of each promoter. The transformants of DSK1 and DSS12 with the recombinant pTH10 and pKT231 plasmid, which has cadA and glnA promoters (each of them is a pressure regulated promoter from DSK1 and DSS12, respectively) and GFP, were grown under high pressure and gene expression of GFP promoted by 50 MPa pressure was confirmed. This is a critical point to create a pressure-sensing bacteria, as the "High Pressure Glow Cells", which will indicate the level of environmental pressure using fluorescence of GFP as a reporter gene.

• 미생물학회지
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• 제35권1호
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• pp.28-34
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• 1999

본 연구에서는 Yeast Artificial Chromosome을 효율적으로 조작하고 유유전자의 기능을 보다 더 용이하게 분석하기 위해 EMCV 바이러스의 IRES 염기서열과 $\beta$-galactosidase를 포함하는 새로운 bicistronic fragmentation vector를 제조하였다. 이 벡터의 폴리클로닝 site에 네 개의희귀한 제한효소 부위를 도입하여 DNA를 용이하게 클로닝할 수 있게 만들었다. 그러므로 원하는 어떤 YAC도 효모세포에서 쉽게 상동 재조함에 의해 절편할 수 있는 장점이 있다. 이 bicistronic fragmentation vector 시스템을 이용하면하나의 메시지로부터 연구하고자 하는 유전자와 $\beta$-galactosidase를 동시에 한 세포에서 발현할 수 있기 때문에 유전자의 발현양상을 쉽게 분석할 수 있는 새로운 도구로 이용할 수 있다.

• Bulletin of the Korean Chemical Society
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• 제33권11호
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• pp.3676-3680
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• 2012

An Epstein-Barr virus (EBV)-based plasmid contains the EBV nuclear antigen 1 (EBNA1) gene and EBV replication origin (oriP) sequence. Since EBNA1 (the only EBV-encoded protein) is combined with oriP, it is replicated simultaneously with chromosomal DNA in human, primate, and canine cells and is faithfully segregated at a stable copy number upon cell division. Consequently, it can be used to stably express gene inserts over a prolonged time in target cells. We have previously shown that the polyamidoamine (PAMAM) dendrimer can be surface-modified with L-arginine. Arginine is present at a high frequency in the transactivator of transcription (Tat) sequences of human immunodeficiency virus (HIV). It presents high membrane permeability and permits effective transfer of DNA inside the cells. In this study, we constructed two kinds of recombinant DNA by inserting the luciferase gene and enhanced green fluorescence protein (eGFP) gene as reporter genes into the pCEP4 plasmid vector. We measured dynamic light scattering (DLS) and zeta potential after preparing PAMAM-based cationic polymer/EBV-based plasmid complexes. We performed transfection of HEK 293 cell lines with the polyplexes, and monitored luciferase activity and green fluorescence protein (GFP) expression. Our results show that PAMAM-based cationic polymer/EBV plasmid complexes provide enhanced and sustained gene expression.

• IMMUNE NETWORK
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• 제13권2호
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• pp.63-69
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• 2013

IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membranebound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-${\alpha}$. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the $CD8^+$ T cell-depleted mice than in $CD4^+$ T cell-depleted or normal mice. These results suggest that $CD8^+$ T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and $CD4^+$ helper T cells.

• Animal Bioscience
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• 제35권2호
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• pp.155-165
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• 2022

Objective: This study was performed to examine whether the porcine glutamic-oxaloacetic transaminase 1 (GOT1) gene has important functions in regulating adipocyte differentiation. Methods: Porcine GOT1 knockout and overexpression vectors were constructed and transfected into the mouse adipogenic 3T3-L1 cells. Lipid droplets levels were measured after 8 days of differentiation. The mechanisms through which GOT1 participated in lipid deposition were examined by measuring the expression of malate dehydrogenase 1 (MDH1) and malic enzyme (ME1) and the cellular nicotinamide adenine dinucleotide phosphate (NADPH) content. Results: GOT1 knockout significantly decreased lipid deposition in the 3T3-L1 cells (p<0.01), whereas GOT1 overexpression significantly increased lipid accumulation (p<0.01). At the same time, GOT1 knockout significantly decreased the NADPH content and the expression of MDH1 and ME1 in the 3T3-L1 cells. Overexpression of GOT1 significantly increased the NADPH content and the expression of MDH1 and ME1, suggesting that GOT1 regulated adipocyte differentiation by altering the NADPH content. Conclusion: The results preliminarily revealed the effector mechanisms of GOT1 in regulating adipose differentiation. Thus, a theoretical basis is provided for improving the quality of pork and studies on diseases associated with lipid metabolism.

• BMB Reports
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• 제46권8호
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• pp.404-409
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• 2013

Extracellular superoxide dismutase (EC-SOD) is a metallo-protein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white.

• Journal of Microbiology and Biotechnology
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• 제34권4호
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• pp.804-811
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• 2024

Foamy viruses (FVs) are generally recognized as non-pathogenic, often causing asymptomatic or mild symptoms in infections. Leveraging these unique characteristics, FV vectors hold significant promise for applications in gene therapy. This study introduces a novel platform technology using a pseudo-virus with single-round infectivity. In contrast to previous vector approaches, we developed a technique employing only two vectors, pcHFV lacking Env and pCMV-Env, to introduce the desired genes into target cells. Our investigation demonstrated the efficacy of the prototype foamy virus (PFV) dual-vector system in producing viruses and delivering transgenes into host cells. To optimize viral production, we incorporated the codon-optimized Env (optEnv) gene in pCMV-Env and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at the 3' end of the transgene in the transfer vector. Consequently, the use of optEnv led to a significant enhancement in transgene expression in host cells. Additionally, the WPRE exhibited an enhancing effect. Furthermore, the introduced EGFP transgene was present in host cells for a month. In an effort to expand transgene capacity, we further streamlined the viral vector, anticipating the delivery of approximately 4.3 kbp of genes through our PFV dual-vector system. This study underscores the potential of PFVs as an alternative to lentiviruses or other retroviruses in the realm of gene therapy.

• Asian-Australasian Journal of Animal Sciences
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• 제26권6호
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• pp.795-803
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• 2013

Various endometrial genes in ruminant ungulates are regulated by conceptus interferon tau (IFNT). However, the effect of each IFNT isoform has not been carefully evaluated. In this study, the effects of 2 IFNT isoforms, paralogs found in utero, and interferon alpha (IFNA) on uterine epithelial and Mardin-Darby bovine kidney (MDBK) cells were evaluated. Expression vectors of the bovine interferon (bIFNT) genes bIFNT1, bIFNTc1, and bIFNA were constructed, and recombinant bIFNs (rbIFNs) were produced by 293 cells. Bovine uterine epithelial or MDBK cells were cultured in the presence or absence of increasing concentrations of each rbIFN for 24, 48, or 72 h. Transcript levels of the IFN-stimulated genes (ISGs) ISG12, ISG15, MX1, and MX2 were analyzed using quantitative reverse transcription-polymerase chain reaction. These messenger RNAs were up-regulated by rbIFN in a time- and concentration-dependent manner. In the epithelial cells, the ISG12 transcript level increased at 48 h after rbIFN treatment but slightly decreased at 72 h, whereas the transcript level of ISG15 increased at 24 h and was maintained through 72 h. Expressions of MX1 and MX2 increased at 72 h after rbIFN treatment. MX1 expression increased in all treatment groups, but MX2 increased only by bIFNTc1. In MDBK cells, the expression of ISG12 was increased by bIFNT1 and bIFNTc1 after 24 and 72 h; however, it was unchanged by rbIFNA. ISG15 increased following the same pattern as that seen in uterine epithelial cells, and MX1 showed a similar expression pattern. MX2 expression was increased by bIFNTc1 treatment in uterine epithelial cells, and its expression was increased by both bIFNT1 and bIFNTc1 in MDBK cells. These results show that epithelial and MDBK cell responses to IFNs differ, suggesting that IFNs possess common functions, but may have acquired different functions following gene duplication.

• 대한수의학회지
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• 제52권3호
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• pp.183-191
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• 2012

The maintenance of peripheral immune tolerance and prevention of chronic inflammation and autoimmune disease require $CD4^{+}CD25^{+}$ T cells (regulatory T cells). The transcription factor Foxp3 is essential for the development of functional, regulatory T cells, which plays a prominent role in self-tolerance. Retroviral vectors can confer high level of gene transfer and transgene expression in a variety of cell types. Here we observed that following retroviral vector-mediated gene transfer of Foxp3, transductional Foxp3 expression was increased in the liver, lung, brain, heart, muscle, spinal cord, kidney and spleen. One day after vector administration, high levels of transgene and gene expression were observed in liver and lung. At 2 days after injection, transductional Foxp3 expression level was increased in brain, heart, muscle and spinal cord, but kidney and spleen exhibited a consistent low level. This finding was inconsistent with the increase in both $CD4^{+}CD25^{+}$ T cell and $CD4^{+}Foxp3^{+}$ T cell frequencies observed in peripheral immune cells by fluorescence-activated cell-sorting (FACS) analysis. Retroviral vector-mediated gene transfer of Foxp3 did not lead to increased numbers of $CD4^{+}CD25^{+}$ T cell and $CD4^{+}Foxp3^{+}$ T cell. These results demonstrate the level and duration of transductional Foxp3 gene expression in various tissues. A better understanding of Foxp3 regulation can be useful in dissecting the cause of regulatory T cells dysfunction in several autoimmune diseases and raise the possibility of enhancing suppressive functions of regulatory T cells for therapeutic purposes.