• Animal cells and systems
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• v.15 no.2
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• pp.123-130
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• 2011

Transient receptor potential melastatin 7 (TRPM7) channels are novel $Ca^{2+}$-permeable non-selective cation channels that are ubiquitously expressed. Activation of TRPM7 channels has been shown to be involved in the survival of gastric cancer cells. Here we show evidence suggesting that TRPM7 channels play an important role in $Zn^{2+}$- mediated cellular injury. Using a combination of electrophysiology, pharmacological analysis, small interfering RNA (siRNA) methods and cell death assays, we showed that activation of TRPM7 channels augmented $Zn^{2+}$-induced apoptosis of AGS cells, the most common human gastric adenocarcinoma cell line. The $Zn^{2+}$-mediated cytotoxicity was inhibited by the non-specific TRPM7 blockers $Gd^{3+}$ or 2 aminoethoxydiphenyl borate (2-APB) and TRPM7 specific siRNA. In addition, we showed that overexpression of TRPM7 channels in HEK293 cells increased $Zn^{2+}$- induced cell injury. Thus, TRPM7 channels may represent a novel target for physiological disorders where $Zn^{2+}$ toxicity plays an important role.

• Journal of Microbiology and Biotechnology
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• v.30 no.12
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• pp.1919-1926
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• 2020

CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.

• Biomedical Science Letters
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• v.19 no.2
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• pp.90-97
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• 2013

The tuberculin skin test (TST) and interferon gamma (IFN-${\gamma}$) release assay (IGRA) have been widely used for diagnosis of latent tuberculosis infection (LTBI). In order to overcome limitations of current LTBI diagnostic methods, the development of a novel molecular assay which is able to measure the IFN-${\gamma}$ messenger RNA (mRNA) expression level after stimulation with Mycobacterium tuberculosis (MTB) specific antigen was recently developed. The ability of a molecular assay to detect MTB infection was similar to commercial IGRA however, the optimal incubation time for stimulating IFN-${\gamma}$ was not yet established. Therefore, in this study the direct comparisons of MTB Ag stimulation times (4 and 24 hrs) were performed for diagnosis of MTB infection. Data showed that the coincident rate between QFT-GIT IFN-${\gamma}$ ELISA and IFN-${\gamma}$ RT-PCR (4 hrs) was 88.35% and that of QFT-GIT and IFN-${\gamma}$ RT-PCR (24 hrs) was 70.85%. Based on a receiver operating characteristic (ROC) curve, the 4 hrs-MTB specific Ag stimulation time for IFN-${\gamma}$ RT-PCR had the significant P value, 95% CI value, and AUC (P < 0.0001, 95% CI=0.82 to 1.02, and AUC=0.9214) in comparison with 24 hrs-MTB specific Ag stimulation time (P = 0.009, 95% CI=0.06 to 0.94, and AUC=0.7711). These results show that 4-hr was the most optimal MTB Ag stimulation time for performing IFN-${\gamma}$ RT-PCR. Although semi-quantitative RT-PCR had a few analytical limitations, it might be useful as an alternative molecular diagnostic method for detecting MTB infection.

• IMMUNE NETWORK
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• v.3 no.3
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• pp.219-226
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• 2003

Background: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than $10^9$ members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to $10^{14}$ molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. Methods: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. Results: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. Conclusion: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.186-192
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• 2008

For the development of specific and effective basic genetic materials to inhibit replication of hepatitis C virus (HCV), HCV genome-targeting trans-splicing aptazyme, which activity is allosterically regulated by a specific ligand, was developed. The aptazyme was designed to be comprised of sequence of RNA aptamer to the ligand, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding the ligand to the aptamer, and trans-splicing ribozyme targeting +199 nt of HCV IRES. Especially, when the aptamer and the communication module was inserted at both P6 and P8 catalytic domain of the specific ribozyme, allosteric activity of the aptazyme was the most induced. The aptazyme was shown to induce activity of trans-splicing reaction specifically and efficiently only in the presence of the specific ligand, but neither in the absence of any ligand nor in the presence of control ligand. This aptazyme can be used as a specific and effective genetic agent against HCV, and a tool for the isolation of anti-HCV lead compounds.

• KSBB Journal
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• v.18 no.4
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• pp.329-334
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• 2003

The present study was aimed at investigating the regulatory mechanism in tissue-specific expression of insulin-like growth factor-I (IGF-I) gene. The expression of IGF-I gene was determined by a solution hybridization/RNase protection assay using total RNA prepared from rat liver or brain of various ages. The levels of IGF-I transcripts were increased in liver gradually after birth, but decreased in brain. By using an oligonucleotide (FRE) corresponding to the C/EBP binding site of the rat IGF-I exon 1, multiple forms of C/EBP${\alpha}$ and C/EBP${\beta}$ proteins, which have DNA-binding activity, were detected in the rat liver or brain. Western immunoblot and southwestern analyses show that p42$\^$C/EBP${\alpha}$/, p38$\^$C/EBP${\alpha}$/, p35$\^$C/EBP${\alpha}$/, p38$\^$C/EBP${\beta}$/, and p35$\^$C/EBP${\beta}$ form specific complexes with the IGF-I exon 1 oligonucleotide in liver nuclear extract and that p42$\^$C/EBP${\alpha}$/ and p38$\^$C/EBP${\beta}$/ form complexes in brain. These data suggest that the formation of FRE-C/EBP isoform complexes may play important roles in the tissue-specific regulation of IGF-I gene expression.

• Journal of Food Hygiene and Safety
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• v.35 no.6
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• pp.535-543
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• 2020

In addition to the ubiquitous roles of cellular RNA in genetic regulations, gene expression and phenotypic variations in response to environmental cues and chemotactic signals, the regulatory roles of a new type of RNA called extracellular RNAs (exRNAs) are an up-and-coming area of research interest. exRNA is transported outside the cell through membrane blebs known as membrane vesicles or extracellular vesicles (EVs). EV formation is predominant and conserved among all microbial forms, including prokaryotes, eukaryotes, and archaea. This review will focus on the three major topics concerning bacterially derived exRNAs, i.e., 1) the discovery of exRNA and influence of extraneous RNA over bacterial gene regulations, 2) the known secretion mechanism for the release of exRNA, and 3) the possible applications that can be devised with these exRNA secreted by different gram-negative and gram-positive bacteria. Further, this review will also provide an opinion on exRNA- and EV-derived applications such as the species-specific exRNA markers for diagnostics and the possible roles of exRNA in probiotics and the epigenetic regulations of the gut microbiome.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.184-193
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• 2007

In mammalian ovary, steroidogenic acute regulatory (StAR) protein mediates the true rate-limiting step of transport of cholesterol from outer to inner mitochondrial membrane. Appropriate expression of StAR gene represents an indispensable component of steroidogenesis and its regulation has been found to be species specific. However, limited information is available regarding StAR gene expression during estrous cycle in buffalo ovary. In the present study, expression, localization and hormonal regulation of StAR mRNA were analyzed by semi-quantitative RT-PCR in buffalo ovary and partial cDNA was cloned. Total RNA was isolated from whole follicles of different sizes, granulosa cells from different size follicles and postovulatory structures like corpus luteum and Corpus albicans. Semi-quantitative RT-PCR analyses showed StAR mRNA expression in the postovulatory structure, corpus luteum. No StAR mRNA was detected in total RNA isolated from whole follicles of different size including the preovulatory follicle (>9 mm in diameter). However, granulosa cells isolated from preovulatory follicles showed the moderate expression of StAR mRNA. To assess the hormonal regulation of StAR mRNA, primary culture of buffalo granulosa cells were treated with FSH (100 ng/ml) alone or along with IGF-I (100 ng/ml) for 12 to 18 h. The abundance of StAR mRNA increased in cells treated with FSH alone or FSH with IGF-I. However, effect of FSH with IGF-I on mRNA expression was found highly significant (p<0.01). In conclusion, differential expression of StAR messages was observed during estrous cycle in buffalo ovary. Also, there was a synergistic action of IGF-I on FSH stimulation of StAR gene.

• Development and Reproduction
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• v.12 no.2
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• pp.159-168
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• 2008

Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain the undifferentiated state of stem cells. However, molecular mechanisms underlying the function of Nanog remain largely unknown. To elucidate the regulatory roles of Nanog involved in maintenance of P19 embryonal carcinoma (EC) stem cells, we transfected three small interfering RNA (siRNA) duplexes targeted against different regions of the Nanog gene into P19 cells. The Nanog siRNA-100 duplexes effectively decreased the expression of Nanog up to 30.7% compared to other two Nanog siRNAs, the Nanog siRNA-400 (67.9 %) and -793 (53.0%). When examined by RT-PCR and real-time PCR, the expression of markers for pluripotency such as Fgf4, Oct3/4, Rex1, Sox1 and Yes was downregulated at 48 h after transfection with Nanog siRNA-100. Furthermore, expression of the ectodermal markers, Fgf5 and Isl1 was reduced by Nanog knockdown. By contrast, the expression of other markers for pluripotency such as Cripto, Sox2 and Zfp57 was not affected by Nanog knockdown at this time. On the other hand, the expression of Lif/Stat3 pathway molecules and of the endoderm markers including Dab2, Gata4, Gata6 and the germ cell nuclear factor was not changed by Nanog knockdown. The results of this study demonstrated that the knockdown of Nanog expression by RNA interference in P19 cells was sufficient to modulate the expression of pluripotent markers involved in the self-renewal of EC stem cells. These results provide the valuable information on potential downstream targets of Nanog and add to our understanding of the function of Nanog in P19 EC stem cells.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.4
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• pp.456-464
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• 2010

RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.