• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.95-105
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• 2005

Transposon-mediated insertional mutagenesis provides one of the most powerful tools for functional studies of genes in higher plants. This project has been performed to develop a large population of insertional mutations, and to construct databases of molecular information on Ds insertion sites in rice. Ultimate goals are to supply genetic materials and information to analyze gene function and to identify and utilize agronomically important genes for breeding purpose. Two strategies have been employed to generate the large scale of transposon population in a Japonica type rice, Dongjin Byeo; 1) genetic crosses between Ac and Ds lines and 2) plant regeneration from seeds carrying Ac and Ds. Our study showed that over 70% of regenerated plants generally carried independent Ds elements and high activity of transposition was detected only during regeneration period. Ds-flanking DNA amplified from leaf tissues of F2 and T1 (or T2) plants have been amplified via TAIL-PCR and directly sequenced. So far, over 65,000 Ds lines have been generated and over 9,500 Ds loci have been mapped on chromosomes by sequence analysis. Database of molecular information on Ds insertion sites has been constructed, and has been opened to the public and will be updated soon at http://www.niab.go.kr. Detailed functional analysis of more than 30 rice mutants has been performed. Several Ds-tagged rice genes that have been selected for functional analysis will be briefly introduced. We expect that a great deal of information and genetic resources of Ds lines would be obtained during the course of this project, which will be shared with domestic and international rice researchers. In addition to the Japonica rice, we have established the tagging system in an rice line of indica genetic background, MGRI079. MGRI079 (Indica/Japonica) was transformed with Agrobacteria carrying Ac and Ds T-DNA vectors. Among transgenic lines, we successfully identified single-copy Ds and Ac lines in MGR1079. These lines were served as ‘starter lines’ to mutagenize Indica genetic background. To achieve rapid, large scale generation of Ds transposant lines, MGR1079 transformants carrying homozygous Ac were crossed with ones with homozygous Ds, and $F_2$seeds were used for plant regeneration. In this year, over 2,000 regeneration plants were grown in the field. We are able to evaluate the tagging efficiency in the Indica genetic background in the fall.

• Biomedical Science Letters
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• v.14 no.1
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• pp.47-53
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• 2008

Hox genes are known to be transcription factors controlling vertebrate pattern formation along the anteroposterior body axis by regulating many target gene expressions during vertebrate embryogenesis. In order to isolate in vivo Hox responsive target genes, ChIP-cloning technique has been applied using Hoxc8 antibody. Here murine embryo of day 11.5 post coitum (E11.5) highly expressing Hoxc8 gene was used after removing head and tail portions where Hoxc8 is rarely expressing. After fixation with formaldehyde, the chromatin DNAs harboring bound proteins were isolated. After sonication, about 0.5- to 1 Kb chromatin DNAs were immunoprecipitated with anti Hoxc8 antibody. After removing the bound proteins with proteinase K, DNAs were isolated, cloned into the pBluescsript II SK vector, and then sequenced. Total 33 random clones sequenced were anlalyzed to be located at 12 different genomic regions. Among these, 8 turned out to be introns and 4 were intergenic regions localized in random chromosomes. The base composition of total cloned genomic sequences (6608 bp) were AT-rich, i.e., 40% GC. When the Hoxc8 core binding sites, such as TAAT, ATTA, TTAT, and ATAA were analyzed total number of 55, 45, 54, and 55 were found, respectively, which are than twice as many as expected number of 26. Although this in silico analysis does not mean that the ChIP-cloned sequence is real Hoxc8 regulatory element in vivo, these results strongly imply that the DNA fragments cloned through chromatin immunoprecipitation could be very much likely the putative Hoxc8 downstream target genes.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.483-493
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• 2010

Knock-out of a gene by insertional mutagenesis is a direct way to address its function through the mutant phenotype. Among ca. 15,000 gene-trapped Ds insertion lines of rice, we identified one line from selected sensitive lines in highly salt stress. We conducted gene tagging by TAIL-PCR, and DNA gel blot analysis from salt sensitive mutant. A gene encoding an oligopeptide transporter (OPT family) homologue was disrupted by the insertion of a Ds transposon into the OsOPT10 gene that was located shot arm of chromosome 8. The OsOPT10 gene (NP_001062118.) has 6 exons and encodes a protein (752 aa) containing the OPT family domain. RT-PCR analysis showed that the expression of OsOPT10 gene was rapidly and strongly induced by stresses such as high-salinity (250 mM), osmotic, drought, $100\;{\mu}M$ ABA. The subcellular localization assay indicated that OsOPT10 was localized specifically in the plasma membrane. Overexpression of OsOPT10 in Arabidopsis thaliana and rice conferred tolerance of transgenic plants to salt stress. Further we found expression levels of some stress related genes were inhibited in OsOPT10 transgenic plants. These results suggested that OsOPT10 might play crucial but differential roles in plant responses to various abiotic stresses.

• Molecules and Cells
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• v.40 no.8
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• pp.567-576
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• 2017

The $Na^+/H^+$ exchanger is responsible for maintaining the acidic tumor microenvironment through its promotion of the reabsorption of extracellular $Na^+$ and the extrusion of intracellular $H^+$. The resultant increase in the extracellular acidity contributes to the chemoresistance of malignant tumors. In this study, the chemosensitizing effects of cariporide, a potent $Na^+/H^+-exchange$ inhibitor, were evaluated in human malignant mesothelioma H-2452 cells preadapted with lactic acid. A higher basal level of phosphorylated (p)-AKT protein was found in the acid-tolerable H-2452AcT cells compared with their parental acid-sensitive H-2452 cells. When introduced in H-2452AcT cells with a concentration that shows only a slight toxicity in H-2452 cells, cariporide exhibited growth-suppressive and apoptosis-promoting activities, as demonstrated by an increase in the cells with pyknotic and fragmented nuclei, annexin V-PE(+) staining, a $sub-G_0/G_1$ peak, and a $G_2/M$ phase-transition delay in the cell cycle. Preceding these changes, a cariporide-induced p-AKT down-regulation, a p53 up-regulation, an ROS accumulation, and the depolarization of the mitochondrial-membrane potential were observed. A pretreatment with the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 markedly augmented the DNA damage caused by the cariporide, as indicated by a much greater extent of comet tails and a tail moment with increased levels of the p-histone H2A.X, $p-ATM^{Ser1981}$, $p-ATR^{Ser428}$, $p-CHK1^{Ser345}$, and $p-CHK2^{Thr68}$, as well as a series of pro-apoptotic events. The data suggest that an inhibition of the PI3K/AKT signaling is necessary to enhance the cytotoxicity toward the acidtolerable H-2452AcT cells, and it underlines the significance of proton-pump targeting as a potential therapeutic strategy to overcome the acidic-microenvironment-associated chemotherapeutic resistance.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.77-77
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• 2003

Chimeric animals are referred to as an organism composed of tissues derived from more than one species. In order to examine if a pluripotency of embryonic stem cells can cross the limitation of a species, we tried to establish human-mouse chimeric animals. Human embryonic stem cells were genetically modified to express eGFP using eukaryonic expression vector pcDNA 3.1 (In Vitrogene) for an easy identification. After selection with neomycin, approximately 15 cells were implanted into mouse blastocoele cavity. Ten chimeric blastocysts were transferred to one of the uterine horn of 2.5 days pesudopregnent ICR female. Out of 272 blastocysts transferred to pseudopregnant recipients 20 live newborn were obtained after 20 days. When newborn were obtained, pups were quickly removed immersed into 4% PFA. By histological examination using fluorescent microscope, green fluorescence was observed from the liver, heart, and spleen in newborn mice. Three weeks after born, presence of eGFP sequence within mouse genome (tail and kidney) was reconfirmed by PCR. eGFP sequence was amplified from the progenies of the animal suggesting a genetic transmission of the transgene. These chimeric mice having human cells at the beginning of development, are expected to recognize human cells as “self”, therefore, human cells or tissues will be able to escape the immunological surveillance of the host if grafted into the animal. These animals will serve as a good model system for studying the graft rejection in tissue transplantation and the potential of the cells to work well in many human disease.

• Journal of Animal Science and Technology
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• v.46 no.6
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• pp.897-902
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• 2004

To improve the methods used for the identification of Hanwoo, we performed a PCR using 3 -tailed primer associated with single nucleotide polymorphism(SNP) in Melanocortin 1 receptor(MCIR) gene. MCIR plays an important role in melanin synthesis and the SNP within MCIR was used as a target for PCRRFLP studies previously. A forward 3 -tailed primer, which matches with the template DNA of Hanwoo but not with others(blackhaired; Holstein and Black angus) at the site of 594th base sequence, and one reverse primer were designed for this study. When use this primer set, a size of 343bp was amplified by PCR only in Hanwoo, not in Holstein and Black angus. This result suggests that the PCR using our 3 -tailed primer would be very accurate, easy, reproducible and economic method to discriminate between Hanwoo meat and other black-haired ones.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.696-703
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• 2019

Cronobacter sakazakii is an opportunistic pathogen causing serious infections in neonates. In this study, a bacteriophage ${\Phi}CS01$, which infects C. sakazakii, was isolated from swine feces and its morphology, growth parameters, and genomic analysis were investigated. Transmission electron microscopy revealed that ${\Phi}CS01$ has a spherical head and is 65.74 nm in diameter with a 98.75 nm contracted tail, suggesting that it belongs to the family Myoviridae. The major viral proteins are approximately 71 kDa and 64 kDa in size. The latent period of ${\Phi}CS01$ was shown to be 60 min, and the burst size was 90.7 pfu (plaque-forming units)/infected cell. Bacteriophage ${\Phi}CS01$ was stable at $4-60^{\circ}C$ for 1 h and lost infectivity after 1 h of heating at $70^{\circ}C$. Infectivity remained unaffected at pH 4-9 for 2 h, while the bacteriophage was inactivated at pH <3 or >10. The double-stranded ${\Phi}CS01$ DNA genome consists of 48,195 base pairs, with 75 predicted open reading frames. Phylogenetic analysis is closely related to that of the previously reported C. sakazakii phage ESP2949-1. The newly isolated ${\Phi}CS01$ shows infectivity in the host bacterium C. sakazakii, indicating that it may be a promising alternative to antibacterial agents for the removal of C. sakazakii from powdered infant formulas.

• Fisheries and Aquatic Sciences
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• v.21 no.12
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• pp.37.1-37.10
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• 2018

The larvae and juveniles of Porocottus leptosomus belonging to the family Cottidae were collected (n = 95, 3.9-16.5 mm in body length, BL) from Busan, Korea, in March 2015. The larvae and juvenile were identified using DNA barcoding as P. leptosomus, and their morphological description was presented in detail. The yolk-sac larvae (3.9-5.6 mm BL) body was slightly compressed, the head was large, the eye was round and large, and the anus was before the middle of the body. The preflexion larvae (5.2-10.0 mm BL) body length drastically increased; caudal fin rays began to occur. The flexion larvae (9.4-11.8 mm BL) notochord flexion started; dorsal, pectoral, and anal fin rays began to occur; pelvic fin buds are seen; they possessed a pair of parietal spine; and a pair of supraocular cirri was first to develop. At 12 mm BL, the notochord was completely flexed. The larva stage (3.9-12.6 mm SL) had the stellate melanophores in the head, isthmus, gut, and tail (along to the ventral midline). During the juvenile stage (11.4-16.5 mm BL), melanophores covered the head and began to form five black bands on the side of the body. The larvae of P. leptosomus spent pelagic life, but moved to the bottom during the juvenile stage. The larvae and juveniles of P. leptosomus differ from other cottid larval fishes by body shape, melanophore head pattern, and spine development. P. leptosomus can be distinguished from Porocottus allisi by morphological development and the occurrence of larval fish: preopercular spine development, melanophore pattern, and caudal fin development.

• Journal of Radiation Industry
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• v.10 no.4
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• pp.211-217
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• 2016

We examined damages from gamma-irradiaion and determined the optimal gamma-ray dose for mutation breeding in lentil (Lens culinaris L.) bean. Four individual lines (L-C, L-2, L-8 and L-9), that have remarkable adaptability in South Korea were gamma-irradiated at doses of 50, 70, 100, 200, 300, 400, and 500 Gy. The germination rate of seed decreased as the dose increased over 50 Gy in all lines. However, $LD_{50}$ and $RD_{50}$ were different among lines. The median lethal doses($LD_{50}$) were approximately 127 (L-C), 74 (L-2), 95 (L-8), and 144 (L-9) Gy. The median reduction doses($RD_{50}$) for plant height, number of leaves, root length, and flash weight were 156, 176, 150, and 180 Gy for L-C, 253, 198, 127, and 142 Gy for L-2, 188, 175, 200, and 190 Gy for L-8, and 162, 210, 224, and 184 for L-9, respectively. The growth characteristics of the $M_1$ generation decreased as the dose increased over 70 Gy. The optimal doses of gamma irradiation for mutation breeding of lentil were determined to be 70 Gy (L-2, L-8) and 100 Gy (L-C, L-9). We performed the comet assay to observe nuclear DNA damage induced by gamma-irradiation. In comet assay, a clear difference was identified over 100 Gy treatments. With increasing doses of gamma-ray in the range of 50 to 500 Gy, the rate of head DNA was decreased significantly from 97.5% to 81.6%. Tail length was consecutively increased from $1.9{\mu}m$ to $17.4{\mu}m$. Our result provides basic information for construction of mutant pools in lentils.

• Parasites, Hosts and Diseases
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• v.57 no.1
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• pp.9-15
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• 2019

Melting temperature shift ($T_m-shift$) is a new detection method that analyze the melting curve on real-time PCR thermocycler using SYBR Green I fluorescent dye. To establish a $T_m-shift$ method for the detection of Ancylostoma ceylanicum and A. tubaeforme in cats, specific primers, with GC tail of unequal length attached to their 5' end, were designed based on 2 SNP loci (ITS101 and ITS296) of the internal transcribed spacer 1 (ITS1) sequences. The standard curve of $T_m-shift$ was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The $T_m-shift$ method stability, sensitivity, and accuracy were tested with reference to the standard curve, and clinical fecal samples were also examined. The results demonstrated that the 2 sets of primers based on the 2 SNPs could accurately distinguish between A. ceylanicum and A. tubaeforme. The coefficient of variation (CV) of $T_m$- values of AceP and AtuP was 0.07% and 0.06% in ITS101 and was 0.06% and 0.08% in ITS296, respectively. The minimum detectable DNA concentration was $5.22{\times}10^{-6}$ and $5.28{\times}10^{-6}ng/{\mu}l$ samples of AceP and AtuP, respectively. The accuracy of $T_m-shift$ method reached 100% based on examination of 10 hookworm DNA samples with known species. In the clinical detection of hookworm in 69 stray cat fecal sample, the $T_m-shift$ detection results were consistent with the microscopic examination and successfully differentiated between the 2-hookworm species. In conclusion, the developed method is a rapid, sensitive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.