• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.29-29
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• 2003

Incompatible plant-pathogen interactions result in the rapid cell death response known as hypersensitive response (HR) and activation of host defense-related genes. To understand the molecular and cellular mechanism controlling defense response better, several approaches including isolation and characterization of novel genes, promoter analysis of those genes, protein-protein interaction analysis and reverse genetic approach etc. By using the yeast two-hybrid system a clone named Tsipl, Tsil -interacting protein 1, was isolated whose translation product apparently interacted with Tsil, an EREBP/AP2 type DNA binding protein. RNA gel blot analysis showed that the expression of Tsipl was increased by treatment with NaCl, ethylene, salicylic acid, or gibberellic acid. Transient expression analysis using a Tsipl::smGFP fusion gene in Arabidopsis protoplasts indicated that the Tsipl protein was targeted to the outer surface of chloroplasts. The targeted Tsipl::smGFP proteins were diffused to the cytoplasm of protoplasts in the presence of salicylic acid (SA) The PEG-mediated co-transfection analysis showed that Tsipl could interact with Tsil in the nucleus. These results suggest that Tsipl-Tsil interaction might serve to regulate defense-related gene expression. Basically the useful promoters are valuable tools for effective control of gene expression related to various developmental and environmental condition.(중략)

• Fisheries and Aquatic Sciences
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• v.12 no.2
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• pp.98-103
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• 2009

The potential utility of the rockbream (Oplegnathus fasciatus) $\beta$-actin 5'-upstream sequence as a regulatory element for DNA vaccination was evaluated based on in vitro and in vivo heterologous expression assays. In the in vitro transfection experiment, the efficacy of the rockbream $\beta$-actin promoter to drive the expression of a downstream lacZ gene was significantly higher (more than fourfold) than that of the human cytomegalovirus (hCMV) promoter in two fish cell lines (grunt Haemulon plumierii fin and bluegill Lepomis macrochirus fry cell lines). In contrast, the functional activity of the rockbream $\beta$-actin promoter was hardly detectable in a mammalian mouse embryonic fibroblast cell line. Rockbream skeletal muscles injected in vivo with a GFP reporter construct driven by the $\beta$-actin promoter displayed the significantly higher expression of a GFP protein (more than threefold) than did those injected with hCMV promoter driven construct. Data from this study suggest that the homologous rockbream $\beta$-actin promoter could be used as a potential regulator for DNA vaccination in this species.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.5
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• pp.621-628
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• 2012

The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000$^{TM}$. The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.98-98
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• 2003

Direct gene transfer to mammalian tissues has significant potential for gene therapy and transgenesis. Liposome-mediated in vivo transfection has begun to gain attention as an alternative to viral vectors, and may also be a good mode of transfection in gene transfer. Interestingly, polymerized cationic liposomes are reported to be very stable in the bloods and efficient for in vivo gene transfer. To examine a possible gene delivery in vivo, we investigated the efficacy and safety of the liposome-mediated gene transfer using vein injection in chick or mouse as model animals. The number of injected pGFP-LacZ using either a commercial or home-made liposomes was 8 and 19 at 16 and 7 day of hatch, respectively. One of injected chick of each experiments was analyzed and the rest is being bred. In mouse, 4/22 showed expression of pGFP-LacZ but 8/22 showed no expression and the remaining animals are also being bred. After injection of liposome/pGFP-LacZ complex into wing vein of 7 or 16 day-old chick, pGFP-LacZ was detected in various tissues isolated from not only young chick but also old chick were turned out to possess. exogenous DNA. Transcripts and proteins of the transgene were also detected by RT-PCR or histochemical analysis, respectively. These results suggest that injected DNA were inserted to genome and produced mRNA and proteins in various tissues and may give an important tools for effective gene delivery in gene therapy or transgenesis.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.91-94
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• 2000

The suspension cells of Taxus cuspidata were transformed with Agrobacterium tumefaciens harboring binary vector pCAMBIE1302 encoding mgfp. Transient transfection efficiency was compared by using the fluoremetric measurement. The transient transfection efficiency was improved by transformation with DMSO and/or sonication treatment. Optimum conditions for DMSO and sonication treatment were 3% and 30sec, respectively. selection and maintenance of transformed cells were continued for 3 months. An insertion of the mgfp gene in transformed cells was detected by PCR and an expression of GFP confirmed by the western blot analysis.

• Bulletin of the Korean Chemical Society
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• v.30 no.9
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• pp.1981-1984
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• 2009

Subcellular localization of protein kinase often plays an important role in determining its activity and specificity. Protein kinase C (PKC), a family of multi-gene protein kinases has long been known to be translocated to the particular cellular compartments in response to DAG or its analog phorbol esters. We used C-terminal green fluorescent protein (GFP) fusion proteins of PKC isoforms to visualize the subcellular distribution of individual PKC isoforms. Intracellular localization of PKC-GFP proteins was monitored by fluorescence microscopy after transient transfection of PKC-GFP expression vectors in the HeLa cells. In unstimulated HeLa cells, all PKC isoforms were found to be distributed throughout the cytoplasm with a few exceptions. PKC$\theta$ was mostly localized to the Golgi, and PKC$\gamma$, PKC$\delta$ and PKC$\eta$ showed cytoplasmic distribution with Golgi localization. DAG analog TPA induced translocation of PKC-GFP to the plasma membrane. PKC$\alpha$, PKC$\eta$ and PKC$\theta$ were also localized to the Golgi in response to TPA. Only PKC$\delta$ was found to be associated with the nuclear membrane after transient TPA treatment. These results suggest that specific PKC isoforms are translocated to different intracellular sites and exhibit distinct biological effects.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.204-204
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• 2022

Transformant construction using protoplasts requires less sample preparation time than particle bombardment and Agrobacterium-mediated transfection. There are two protoplast transfection methods: the PEG-mediated transfection method and the Lipofectamine transfection method. When Lipofectamine is mixed with DNA, Lipofectamine surrounds DNA like a cell membrane because of the positive charge of Lipofectamine. The Lipofectamine-DNA complex makes DNA insertion into cells easier. Fectin has similar functions to lipofectamine and is less expensive than lipofectamine. The 3D-fectin technology has been highlighted in animal cell transfection. Therefore, we performed PEG-mediated transfection, Lipofectamine transfection, and 3D-pectin transfection with a GFP construct. Protoplasts were isolated using the first leaf of "Bobwhite" after 4 hours of incubation in an isolation Buffer (cellulase + macerozyme). Protoplasts transformed by each method were cultured for 48 hours, and then GFP fluorescence expression was confirmed under confocal microscopy. GFP signals were detected in PEG-mediated transfection and Lipofectamine transfection. And the GFP signals were also detected in protoplasts to which 3D-fectin technology was applied, suggesting that 3D-fectin technology can be used for plant protoplast transfection.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.431-439
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• 2014

We report the construction of two Bacillus subtilis expression vectors, pBNS1/pBNS2. Both vectors are based on the strong promoter P43 and the ampicillin resistance gene expression cassette. Additionally, a fragment with the Shine-Dalgarno sequence and a multiple cloning site (BamHI, SalI, SacI, XhoI, PstI, SphI) were inserted. The coding region for the amyQ (encoding an amylase) signal peptide was fused to the promoter P43 of pBNS1 to construct the secreted expression vector pBNS2. The applicability of vectors was tested by first generating the expression vectors pBNS1-GFP/pBNS2-GFP and then detecting for green fluorescent protein gene expression. Next, the mannanase gene from B. pumilus Nsic-2 was fused to vector pBNS2 and we measured the mannanase activity in the supernatant. The mannanase total enzyme activity was 8.65 U/ml, which was 6 times higher than that of the parent strain. Our work provides a feasible way to achieve an effective transformation system for gene expression in B. subtilis and is the first report to achieve B. pumilus mannanase secretory expression in B. subtilis.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.797-804
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• 2000

The human immunodeficiency virus type 1 (HIV-1) Tat is one of the viral gene products essential for HIV replication. The exogenous Tat protein is transduced through the plasma membrane and then accumulated in a cell. The basic domain of the Tat protein, which is rich in arginine and lysine residues and called the protein transduction domain (PTD), has been identified to be responsible for this transduction activity. To better understand the nature of the transduction mediated by this highly basic domain of HIV-1 Tat, the Green Fluorescent Protein (GFP) was expressed and purified as a fusion protein with a peptide derived from the HIV-1 Tat basic domain in Escherichia coli. The transduction of Tat-GFP into mammalian cells was then determined by a Western blot analysis and fluorescence microscopy. The cells treated with Tat-GFP exhibited dose- and time-dependent increases in their intracellular level of the protein. the effective transduction of denatured Tat-GFP into both the nucleus and the cytoplasm of mammalian cells was also demonstrated, thereby indicating that the unfolding of the transduced protein is required for efficient transduction. Accordingly, the availability of recombinant Tat-GFP can facilitate the simple and specific identification of the protein transduction mediated by the HIV-1 Tat basic domain in living cells either by fluorescence microscopy or by a fluorescence-activated cell sorter analysis.

• Journal of Microbiology and Biotechnology
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• v.17 no.7
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• pp.1213-1216
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• 2007

The vector pCW5 with plasmid pC7, originally isolated in Lactobacillus paraplantarum C7 derived from kimchi, was constructed using a p32 strong promoter, the pC7 replicon, and green fluorescent protein (GFP) as the reporter. The constructed vector was transformed into E. coli and Leuconostoc mesenteroides, and GFP expression detected using a Western blot analysis. GFP fluorescence was recognized in E. coli and Leuconostoc mesenteroides using a confocal microscope. In addition, GFP fluorescence was also clearly detected in several industrially important lactic acid bacteria (LAB), including Lactobacillus bulgaricus, Lactobacillus paraplantarum, and Lactobacillus plantarum. Thus, pCW5 was shown to be effective for Leuconostoc mesenteroides when using GFP as the reporter, and it can also be used as a broad-host-range vector for other lactic acid bacteria.