• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.273-276
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• 2006

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of lower organisms. Recently, we have reported that a transfection of human U6 promoter (hU6) driven hairpin small-interference RNA (siRNA) plasmid specifically knocks down the target gene by post-transcriptional gene silencing in mammalian cells. Here we report that transfection of polymerase chain reaction (PCR) products, containing human U6 promoter with hairpin siRNA, knocks down the target gene expression in human teratocarcinoma NT-2/D1 cells. Moreover, we showed 3' end termination sequence, 5 Ts, is not critical elements for knocking down in PCR-based siRNA system. Therefore, the PCR-based siRNA system is a promising tool not only for the screening but also to temporally regulate gene expression in the human progenitor cells.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.213-225
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• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• Journal of Integrative Natural Science
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• v.12 no.4
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• pp.142-146
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• 2019

Peroxidasin is a unique member of peroxidase family in that it has extracellular matrix (ECM) motif as well as peroxidase activity. Peroxidasins are involved in consolidation the extracellular matrix during development and in innate immune defense. C. elegans has two functional peroxidasins, PXN-1 and PXN-2, and PXN-2 is known to contribute to innate immune system. However, it is not clear of PXN-1 function in innate immune system. Therefore, this study is focused on the function of PXN-1 and the relationship between PXN-1 and PXN-2 in innate defense system in C. elegans. When pxn-1 was knocked down by RNAi, the worm turned to be more resistant to pathogens, Staphylococcus aureus and Pseudomonas aeruginosa and the enhanced resistance was abolished in pxn-1pxn-2 double knock down. By contrast, pxn-2 knock down showed stronger susceptibility to the pathogens. These results suggest that pxn-2 can contribute the pathogen resistance and pxn-1 can suppress the pathogen resistance. To confirm the idea, overexpression experiments were performed. Overexpression of pxn-1 showed more susceptible to pathogens compared to the control and double overexpression of pxn-1pxn-2 overcame the susceptibility of pxn-1 overexpression to the pathogens. On the other hand, pxn-2 overexpression made the worm more resistant to the pathogens and the resistance was maintained in pxn-1pxn-2 double overexpression. The comparison of the susceptibilities to the bacterial pathogens in above mentioned constructs suggests that PXN-1 suppress the function of PXN-2 in defense against bacterial pathogens in Caenorhabditis elegans.

• BMB Reports
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• v.44 no.9
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• pp.595-600
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• 2011

Pax 6, a member of the paired box (Pax) family, has been implicated in oncogenesis. However, its therapeutic potential has been never examined in breast cancer. To explore the role of Pax6 in breast cancer development, a lentivirus based short hairpin RNA (shRNA) delivery system was used to knockdown Pax6 expression in estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells. Effect of Pax6 silencing on breast cancer cell proliferation and tumorigenesis was analyzed. Pax6-RNAi-lentivirus infection remarkably downregulated the expression levels of Pax6 mRNA and protein in MCF-7 and MDA-MB-231 cells. Accordingly, the cell viability, DNA synthesis, and colony formation were strongly suppressed, and the tumorigenesis in xenograft nude mice was significantly inhibited. Moreover, tumor cells were arrested at G0/G1 phase after Pax6 was knocked down. Pax6 facilitates important regulatory roles in breast cancer cell proliferation and tumor progression, and could serve as a diagnostic marker for clinical investigation.

• BMB Reports
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• v.53 no.9
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• pp.472-477
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• 2020

Osteoclasts are hematopoietic-derived cells that resorb bone. They are required to maintain proper bone homeostasis and skeletal strength. Although osteoclast differentiation depends on receptor activator of NF-κB ligand (RANKL) stimulation, additional molecules further contribute to osteoclast maturation. Here, we demonstrate that protocadherin-7 (Pcdh7) regulates formation of multinucleated osteoclasts and contributes to maintenance of bone homeostasis. We found that Pcdh7 expression is induced by RANKL stimulation, and that RNAi-mediated knockdown of Pcdh7 resulted in impaired formation of osteoclasts. We generated Pcdh7-deficient mice and found increased bone mass due to decreased bone resorption but without any defect in bone formation. Using an in vitro culture system, it was revealed that formation of multinucleated osteoclasts is impaired in Pcdh7-deficient cultures, while no apparent defects were observed in differentiation and function of Pcdh7-deficient osteoblasts. Taken together, these results reveal an osteoclast cell-intrinsic role for Pcdh7 in maintaining bone homeostasis.

• Molecules and Cells
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• v.43 no.6
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• pp.530-538
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• 2020

The Gustatory system enables animals to detect toxic bitter chemicals, which is critical for insects to survive food induced toxicity. Cucurbitacin is widely present in plants such as cucumber and gourds that acts as an anti-herbivore chemical and an insecticide. Cucurbitacin has a harmful effect on insect larvae as well. Although various beneficial effects of cucurbitacin such as alleviating hyperglycemia have also been documented, it is not clear what kinds of molecular sensors are required to detect cucurbitacin in nature. Cucurbitacin B, a major bitter component of bitter melon, was applied to induce action potentials from sensilla of a mouth part of the fly, labellum. Here we identify that only Gr33a is required for activating bitter-sensing gustatory receptor neurons by cucurbitacin B among available 26 Grs, 23 Irs, 11 Trp mutants, and 26 Gr-RNAi lines. We further investigated the difference between control and Gr33a mutant by analyzing binary food choice assay. We also measured toxic effect of Cucurbitacin B over 0.01 mM range. Our findings uncover the molecular sensor of cucurbitacin B in Drosophila melanogaster. We propose that the discarded shell of Cucurbitaceae can be developed to make a new insecticide.

• Molecules and Cells
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• v.40 no.1
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• pp.73-81
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• 2017

The ${\gamma}$-secretase complex represents an evolutionarily conserved family of transmembrane aspartyl proteases that cleave numerous type-I membrane proteins, including the ${\beta}$-amyloid precursor protein (APP) and the receptor Notch. All known rare mutations in APP and the ${\gamma}$-secretase catalytic component, presenilin, which lead to increased amyloid ${\beta}$-peptide production, are responsible for early-onset familial Alzheimer's disease. ${\beta}$-amyloid protein precursor-like (APPL) is the Drosophila ortholog of human APP. Here, we created Notch- and APPL-based Drosophila reporter systems for in vivo monitoring of ${\gamma}$-secretase activity. Ectopic expression of the Notch- and APPL-based chimeric reporters in wings results in vein truncation phenotypes. Reporter-mediated vein truncation phenotypes are enhanced by the Notch gain-of-function allele and suppressed by RNAi-mediated knockdown of presenilin. Furthermore, we find that apoptosis partly contributes to the vein truncation phenotypes of the APPL-based reporter, but not to the vein truncation phenotypes of the Notch-based reporter. Taken together, these results suggest that both in vivo reporter systems provide a powerful genetic tool to identify genes that modulate ${\gamma}$-secretase activity and/or APPL metabolism.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• BMB Reports
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• v.56 no.12
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• pp.657-662
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• 2023

Neurodegenerative diseases are characterized by distinct protein aggregates, such as those of α-synuclein and tau. Lysosomal defect is a key contributor to the accumulation and propagation of aberrant protein aggregates in these diseases. The discoveries of common proteinopathies in multiple forms of lysosomal storage diseases (LSDs) and the identification of some LSD genes as susceptible genes for those proteinopathies suggest causative links between LSDs and the proteinopathies. The present study hypothesized that defects in lysosomal genes will differentially affect the propagation of α-synuclein and tau proteins, thereby determining the progression of a specific proteinopathy. We established an imaging-based high-contents screening (HCS) system in Caenorhabditis elegans (C. elegans) model, by which the propagation of α-synuclein or tau is measured by fluorescence intensity. Using this system, we performed RNA interference (RNAi) screening to induce a wide range of lysosomal malfunction through knock down of 79 LSD genes, and to obtain the candidate genes with significant change in protein propagation. While some LSD genes commonly affected both α-synuclein and tau propagation, our study identified the distinct sets of LSD genes that differentially regulate the propagation of either α-synuclein or tau. The specificity and efficacy of these LSD genes were retained in the disease-related phenotypes, such as pharyngeal pumping behavior and life span. This study suggests that distinct lysosomal genes differentially regulate the propagation of α-synuclein and tau, and offer a steppingstone to understanding disease specificity.

• Journal of Microbiology and Biotechnology
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• v.19 no.8
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• pp.765-773
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• 2009

Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur ($Fur_{Et}$) was overproduced, and enhanced when $Fur_{Et}$ was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.