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Development of DNA chip for jellyfish verification from South Korea
Gunsup Lee,박소윤,황진익,Youn-Ho Lee,황승용,이석찬,이택견 한국바이오칩학회 2011 BioChip Journal Vol.5 No.4
Global warming and environmental change have been responsible for an exponential increase in the number of jellyfish. The mass propagation of jellyfish has inflicted great damage to the economy worldwide. Therefore, in order to instantly determine the possible types of jellyfish, DNA barcode data analysis was conducted using molecular markers. DNA chip technology is an efficient method for species-level identification and, as such, will contribute powerfully to taxonomic and biodiversity research. In order to identify jellyfish species, the mitochondrial COI gene, which has been shown to be widely applicable in animal barcoding, was used; the selected species-specific probe was 23 bp and was printed onto silylated slides using a robotic microarrayer. Additionally, COI genes were mplified from the genomic DNA of jellyfish using the primers LCO1490 and HCO2198 targeted to COI. In this study, we amplified and analyzed species-specific COI sequences for six jellyfish species (Aequorea coerulescens, Aurelia aurita, Bolinopsis sp., Cyanea nozakii, Dactylometra quinquecirrha, Nemopilema nomurai) collected at Jangmok Bay in Geoje. Herein, we describe each of the jellyfish species detected via microarray analysis. As a result, the COI barcode sequence technique was found to be suitable for the identification of jellyfish from South Korea.
Lee, Gunsup,Cho, SeungChan,Hoang, Phuong Mai,Kim, Dongjun,Lee, Yongjun,Kil, Eui-Joon,Byun, Sung-June,Lee, Taek-Kyun,Kim, Dae-Hyun,Kim, Sunghan,Lee, Sukchan Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.9
3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and $10{\mu}g$ 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 $LD_{50}$ PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% ($5{\mu}g$) and 47% ($10{\mu}g$). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.
Lee, Gunsup,Choi, Hoonsung,Sureshkumar, Shanmugam,Jung, Sun Keun,Kim, Jeom Sun,Oh, Keon Bong,Kim, Kyung-Woon,Yang, Hyeon,Kim, Dong-Hoon,Byun, Sung June Elsevier 2019 Research in veterinary science Vol.123 No.-
<P><B>Abstract</B></P> <P>Infectious bronchitis (IB) generated by the infectious bronchitis virus (IBV) causes economic difficulties for livestock farmers. The 3D8 single chain variable fragment (scFv) protein is a recombinant antibody with nuclease activity that shows antiviral effects against various DNA and RNA viruses in mice and chickens. In this experiment, 3D8 scFv G<SUB>2</SUB> transgenic chickens produced by crossing 3D8 scFv G<SUB>1</SUB> transgenic rooster and wild type hens were screened by genomic PCR and immunohistochemistry analysis. 3D8 scFv transgenic chickens, wild type sibling chickens, and SPF chickens were directly infected with IBV (5 chickens per group) and indirectly infected by airborne propagation (15 chickens per group). The relative IBV shedding titers were measured by quantitative real-time PCR using oropharyngeal and cloacal swabs on days 3 and 5 after intraocular infection. The viral load was significantly decreased in the 3D8 scFv transgenic chickens from the contact transmission group. Additionally, blood was collected from each group on day 17 post-infection. The ELISA results showed a marked reduction of the antibody titer against IBV in the 3D8 scFv transgenic chickens from the contact transmission group. These results suggest that the 3D8 scFv protein potentially inhibits infectious bronchitis virus transmission in chickens.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Produced G2 3D8 single chain variable fragment (scFv) transgenic chickens. </LI> <LI> 3D8 scFv transgenic chickens showed reduced infectious bronchitis viral shedding level in the contact transmission group. </LI> <LI> 3D8 scFv transgenic chickens were 40% lower than the response in the control groups in IBV serum antibody titer. </LI> </UL> </P>
Lee, Gunsup,Yu, Jaelim,Cho, Seungchan,Byun, Sung-June,Kim, Dae Hyun,Lee, Taek-Kyun,Kwon, Myung-Hee,Lee, Sukchan Public Library of Science 2014 PLoS pathogens Vol.10 No.6
<▼1><P>Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an <I>in vivo</I> mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses <I>in vitro</I> in HeLa cells and in an <I>in vivo</I> mouse system.</P></▼1><▼2><P><B>Author Summary</B></P><P>Most strategies for developing virus-resistant transgenic cells and animals are based on the concept of virus-derived resistance, in which dysfunctional virus-derived products are expressed to interfere with the pathogenic process of the virus in transgenic cells or animals. However, these viral protein targeting approaches are limited because they only target specific viruses and are susceptible to viral mutations. We describe a novel strategy that targets the viral genome itself, rather than viral gene products, to generate virus-resistant transgenic cells and animals. We functionally expressed 3D8 scFv which has both DNase and RNase activities, in HeLa cells and transgenic mice. We found that the transgenic cells and mice acquired complete resistance to two DNA viruses (HSV and PRV) without accumulating the virus, and showed delayed onset of disease symptoms. The antiviral effects against DNA viruses demonstrated in this study were caused by (1) DNase activity of 3D8 scFv in the nucleus, which inhibited DNA replication or RNA transcription and (2) 3D8 scFv RNase activity in the cytoplasm, which blocked protein translation. This strategy may facilitate control of a broad spectrum of viruses, including viruses uncharacterized at the molecular level, regardless of their genome type or variations in gene products.</P></▼2>
Changes in gene expression profile due to acute toxicity of toxaphene in the marine medaka
Lee, Aekyung,Woo, Seonock,Won, Hyokyoung,Lee, Gunsup,Lee, Taek-Kyun,Yum, Seungshic 대한독성유전단백체학회 2013 Molecular & cellular toxicology Vol.9 No.2
Differential gene expression profiling was performed using cDNA microarray hybridization on the hepatic tissue of the marine medaka (Oryzias javanicus) after exposure to toxaphene, which is classified as a persistent organic pollutant. Ninety-seven differentially expressed candidate genes were identified; 40 were induced and 57 were repressed (P<0.05). The genes were assembled into 18 groups based mainly on the Eukaryotic Orthologous Groups classification. These isolated gene candidates were differentially expressed and therefore have great potential as molecular biomarkers for identifying environmental stressors and prognosis for the biological effects of the toxicant. Some of the genes were closely related to endocrine disruption, renal and cardiovascular disease, tumorigenesis, immune responses, and detoxification. Our results will allow future studies to assess the molecular mechanisms of toxaphene toxicity and to develop a systems biology approach to environmental stress biology.