• BMB Reports
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• v.54 no.2
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• pp.118-123
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• 2021

The bacterial effector protein RavZ from a pathogen can impair autophagy in the host by delipidating the mammalian autophagy-related gene 8 (mATG8)-phosphatidylethanolamine (PE) on autophagic membranes. In RavZ, the membrane-targeting (MT) domain is an essential function. However, the molecular mechanism of this domain in regulating the intracellular localization of RavZ in cells is unclear. In this study, we found that the fusion of the green fluorescent protein (GFP) to the MT domain of RavZ (GFP-MT) resulted in localization primarily to the cytosol and nucleus, whereas the GFP-fused duplicated-MT domain (GFP-2xMT) localized to Rab5- or Rab7-positive endosomes. Similarly, GFP fusion to the catalytic domain (CA) of RavZ (GFP-CA) resulted in localization primarily to the cytosol and nucleus, even in autophagy-induced cells. However, by adding the MT domain to GFP-CA (GFP-CA-MT), the cooperation of MT and CA led to localization on the Rab5-positive endosomal membranes in a wortmannin-sensitive manner under nutrient-rich conditions, and to autophagic membranes in autophagy-induced cells. In autophagic membranes, GFP-CA-MT delipidated overexpressed or endogenous mATG8-PE. Furthermore, GFP-CA△α3-MT, an α3 helix deletion within the CA domain, failed to localize to the endosomal or autophagic membranes and could not delipidate overexpressed mATG8-PE. Thus, the CA or MT domain alone is insufficient for stable membrane localization in cells, but the cooperation of MT and CA leads to localization to the endosomal and autophagic membranes. In autophagic membranes, the CA domain can delipidate mATG8-PE without requiring substrate recognition mediated by LC3-interacting region (LIR) motifs.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.5
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• pp.743-748
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• 2017

Objective: Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells. Methods: Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. Results: Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. Conclusion: The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.891-899
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• 2016

The recently isolated aniline-degrading bacterium Pseudomonas migulae AN-1 was tagged with green fluorescent protein (GFP) to investigate its bioaugmentation potential against aniline-contaminated groundwater through microcosm experiments. The survival and cellular response of GFP-tagged AN-1 introduced in a lab-scale aquifer corresponded directly with aniline consumption. During the process, the GFP-tagged AN-1 biomass increased from 7.52 × 10⁵ cells/ml to 128 × 10⁵ cells/ml and the degradation rate of aniline was 6.04 mg/l/h. GFP-tagged AN-1 was moderately hydrophobic (41.74%-47.69%) when treated with 20-100 mg/l aniline and exhibited relatively strong hydrophobicity (55.25%-65.78%) when the concentration of aniline was ≥100 mg/l. The membrane permeability of AN-1 increased followed by a rise in aniline below 100 mg/l and was invariable with aniline above 100 mg/l. Pyrosequencing analysis showed that the relative abundance of Proteobacteria (accounted for 99.22% in the non-bioaugmentation samples) changed to 89.23% after bioaugmentation with GFP-tagged AN-1. Actinobacteria increased from 0.29% to 2.01%, whereas the abundance of Firmicutes barely changed. These combined findings demonstrate the feasibility of removing aniline in aquifers by introducing the strain AN-1 and provide valuable information on the changes in the diversity of dominant populations during bioaugmentation.

• Journal of Korean Neurosurgical Society
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• v.62 no.2
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• pp.153-165
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• 2019

Objective : Spinal cord injury (SCI) is a very serious health problem, usually caused by a trauma and accompanied by elevated levels of inflammation indicators. Stem cell-based therapy is promising some valuable strategies for its functional recovery. Nestin-positive progenitor and/or stem cells (SC) isolated from pancreatic islets (PI) show mesenchymal stem cell (MSC) characteristics. For this reason, we aimed to analyze the effects of rat pancreatic islet derived stem cell (rPI-SC) delivery on functional recovery, as well as the levels of inflammation factors following SCI. Methods : rPI-SCs were isolated, cultured and their MSC characteristics were determined through flow cytometry and immunofluorescence analysis. The experimental rat population was divided into three groups : 1) laminectomy & trauma, 2) laminectomy & trauma & phosphate-buffered saline (PBS), and 3) laminectomy+trauma+SCs. Green fluorescent protein (GFP) labelled rPI-SCs were transplanted into the injured rat spinal cord. Their motilities were evaluated with Basso, Beattie and Bresnahan (BBB) Score. After 4-weeks, spinal cord sections were analyzed for GFP labeled SCs and stained for vimentin, $S100{\beta}$, brain derived neurotrophic factor (BDNF), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), vascular endothelial growth factor (VEGF) and proinflammatory (interleukin [IL]-6, transforming growth factor $[TGF]-{\beta}$, macrophage inflammatory protein [MIP]-2, myeloperoxidase [MPO]) and anti-inflammatory (IL-1 receptor antagonis) factors. Results : rPI-SCs were revealed to display MSC characteristics and express neural and glial cell markers including BDNF, glial fibrillary acidic protein (GFAP), fibronectin, microtubule associated protein-2a,b (MAP2a,b), ${\beta}3$-tubulin and nestin as well as anti-inflammatory prostaglandin E2 receptor, EP3. The BBB scores showed significant motor recovery in group 3. GFP-labelled cells were localized on the injury site. In addition, decreased proinflammatory factor levels and increased intensity of anti-inflammatory factors were determined. Conclusion : Transplantation of PI-SCs might be an effective strategy to improve functional recovery following spinal cord trauma.

• Journal of fish pathology
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• v.23 no.3
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• pp.273-280
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• 2010

To determine whether rock bream Oplegnathus fasciatus can be protected from rock bream iridovirus (RBIV) infection by intramuscular injection of long double-stranded RNAs (dsRNAs), we compared protective effect of virus-specific dsRNAs corresponding to major capsid protein (MCP), ORF 084, ORF 086 genes, and virus non-specific green fluorescent protein (GFP) gene. Furthermore, to determine whether the non-specific type I interferon (IFN) response was associated with protective effect, we estimated the activation of type I IFN response in fish using expression level of IFN inducible Mx gene as a marker. As a result, mortality of fish injected with dsRNAs and challenged with RBIV was delayed for a few days when comparing with PBS injected control group. However, virus-specific dsRNA injected groups exhibited no significant differences in survival period when compared to the GFP dsRNA injected group. Semi-quantitative analysis indicated that the degree of antiviral response via type I IFN response is supposedly equal among dsRNA injected fish. These results suggest that type I IFN response rather than sequence-specific RNA interference might involve in the lengthened survival period of fish injected with virus-specific dsRNAs.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• International Journal of Oral Biology
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• v.32 no.1
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• pp.7-11
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• 2007

It is difficult to introduce DNA in non-invasive manner into oral cancer cells as well as primary cells for gene manipulation and expression in vivo. So far, several methods for a gene delivery have been performed to solve this problem. Magnetofection is one of the recent methods for gene transfer, and nanoparticles are applied under a magnetic field for DNA delivery. We investigated whether the magnetofection increases the efficiency of a gene delivery into several oral cell lines. By using a plasmid coding the green fluorescent protein (GFP), the efficiency of gene transfer by magnetofection was compared with those by using the calcium phosphate and the commercial transfection agent. Indeed, the magnetofection increased the green fluorescent signal in cells, suggested that this method apparently enhance the efficiency of gene delivery without any defects in various oral cancer cell lines. Finally, we have shown that magnetofection can be a useful technique for gene delivery to difficult-to-transfect cells to perform a functional study of genes in vivo.

• Molecules and Cells
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• v.28 no.1
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• pp.25-30
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• 2009

${\beta}$-Arrestins turn off G protein-mediated signals and initiate distinct G protein-independent signaling pathways. We previously demonstrated that angiotensin $AT_1$ receptorbound ${\beta}$-arrestin 1 is cleaved after $Phe^{388}$ upon angiotensin II stimulation. The mechanism and signaling pathway of angiotensin II-induced ${\beta}$-arrestin cleavage remain largely unknown. Here, we show that protein Tyr phosphatase activity is involved in the regulation of ${\beta}$-arrestin 1 cleavage. Tagging of green fluorescent protein (GFP) either to the N-terminus or C-terminus of ${\beta}$-arrestin 1 induced conformational changes and the cleavage of ${\beta}$-arrestin 1 without angiotensin $AT_1$ receptor activation. Orthovanadate and molybdate, inhibitors of protein Tyr phosphatase, attenuated the cleavage of C-terminal GFP-tagged ${\beta}$-arrestin 1 in vitro. The inhibitory effects of okadaic acid and pyrophosphate, which are inhibitors of protein Ser/Thr phosphatase, were less than those of protein Tyr phosphatase inhibitors. Cell-permeable pervanadate inhibited angiotensin II-induced cleavage of ${\beta}$-arrestin 1 in COS-1 cells. Our findings suggest that Tyr phosphorylation signaling is involved in the regulation of angiotensin II-induced ${\beta}$-arrestin cleavage.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.928-933
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• 2007

Cameleon is a genetically engineered $Ca^{2+}$ sensing molecule consisting of two variants of the green fluorescent protein (GFP), calmodulin and calmodulin-binding protein, M13. HEK-293 cells stably expressing three types of cameleons, yellow cameleon-2, cameleon-3er, and cameleon-2nu, were constructed, and the expression and localization of these cameleons were confirmed by fluorescent imaging. Among the cameleons, the yellow cameleon-2 was selected for analyzing the change in $Ca^{2+}$ induced by the olfactory receptor-mediated signal transduction, because it is localized in the cytosol and binds to cytosolic $Ca^{2+}$ ions. Cells stably expressing yellow cameleon-2 were transfected with each of the test olfactory receptor genes, odr-10 and 17, and the expression of the olfactory receptor genes were examined using immunocytochenmical methods and RT-PCR. Stimulating each olfactory receptor with its specific odorant caused an increase in the intracellular $Ca^{2+}$ level, which was measured using yellow cameleon-2. These results demonstrate that yellow cameleon-2 can be conveniently used to examine the function of the olfactory receptors expressed in heterologous cells.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.