• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.273-273
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• 2013

Recently, Point-of-care (POC) testing microdevices enable to do the patient monitoring, drug screening, pathogen detection in the outside of hospital. Immunochromatographic strip (ICS) is one of the diagnostic technologies which are widely applied to POC detection. Relatively low cost, simplicity to use, easy interpretations of the diagnostic results and high stability under any circumstances are representative advantages of POC diagnosis. It would provide colorimetric results more conveniently, if the genetic analysis microsystem incorporates the ICS as a detector part. In this work, we develop a reverse transcriptase-polymerase chain reaction (RT-PCR) microfluidic device integrated with a ROSGENE strip for colorimetric influenza H1N1 virus detection. The integrated RT-PCR- ROSGENE device is consist of four functional units which are a pneumatic micropump for sample loading, 2 ${\mu}L$ volume RT-PCR chamber for target gene amplification, a resistance temperature detector (RTD) electrode for temperature control, and a ROSGENE strip for target gene detection. The device was fabricated by combining four layers: First wafer is for RTD microfabrication, the second wafer is for PCR chamber at the bottom and micropump channel on the top, the third is the monolithic PDMS, and the fourth is the manifold for micropump operation. The RT-PCR was performed with subtype specific forward and reverse primers which were labeled with Texas-red, serving as a fluorescent hapten. A biotin-dUTP was used to insert biotin moieties in the PCR amplicons, during the RT-PCR. The RT-PCR amplicons were loaded in the sample application area, and they were conjugated with Au NP-labeled hapten-antibody. The test band embedded with streptavidins captures the biotin labeled amplicons and we can see violet colorimetric signals if the target gene was amplified with the control line. The off-chip RT-PCR amplicons of the influenza H1N1 virus were analyzed with a ROSGENE strip in comparison with an agarose gel electrophoresis. The intensities of test line was proportional to the template quantity and the detection sensitivity of the strip was better than that of the agarose gel. The test band of the ROSGENE strip could be observed with only 10 copies of a RNA template by the naked eyes. For the on-chip RT-PCR-ROSGENE experiments, a RT-PCR cocktail was injected into the chamber from the inlet reservoir to the waste outlet by the micro-pump actuation. After filling without bubbles inside the chamber, a RT-PCR thermal cycling was executed for 2 hours with all the microvalves closed to isolate the PCR chamber. After thermal cycling, the RT-PCR product was delivered to the attached ROSGENE strip through the outlet reservoir. After dropping 40 ${\mu}L$ of an eluant buffer at the end of the strip, the violet test line was detected as a H1N1 virus indicator, while the negative experiment only revealed a control line and while the positive experiment a control and a test line was appeared.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.275-280
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• 2005

Anthrax is an infectious disease caused by the gram-positive bacterium, Bacillus anthracis. Anthrax toxin is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF onto the cytosol. LF is a zinc-dependent metalloprotease, which is a critical virulence factor in cytotoxicity of infected animals. Therefore, it is of interest to develop its potent inhibitors for the neutralization of anthrax toxin. The first step to identify the inhibitors is the development of a rapid, sensitive, and simple assay method with a high-throughput ability. Much efforts have been concentrated on the preparation of powerful assays and on the screening of inhibitors using these system. In the present study, we have tried to construct anthrax lethal factor in yeast expression system to prepare cell-based high-throughput assay system. Here, we have shown the results covering the construction of a new vector system, subcloning of LF gene, and the expression of target gene. Our results are first trial to express LF gene in eukaryote and provide the basic steps in design of cell-based assay system.

• Korean Journal of Plant Resources
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• v.34 no.4
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• pp.278-286
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• 2021

Efficient in vitro regeneration system is essential for the successful crop breeding of soybean (Glycine max (L.) Merr.) using the new biotechnology. The genotype of donor plants strongly influences the establishment of tissue culture system. Therefore, the screening of genotypes with excellent tissue culture ability is very important for soybean genetic improvement. In this study, we report the tissue culture efficiency of 21 soybean cultivars belong to Korean soybean core-collection and two foreign cultivars (Jack and Maverick). The Kwangan, Anpyeong and Seonam are share close genetic relationship in 21 cultivars and these three cultivars were observed the high frequency of germination and regeneration. Furthermore, the high tissue culture abilities were also observed in the Williams 82 used in reference genome sequencing and the two foreign cultivars. The transformation of pBAtc:tRNA with bar gene was performed by Agrobacterium tumefaciens in the cultivars with high tissue culture ability. Transformation of the bar gene was identified by PCR analysis in Kwangan, Pungwon, Seonam, and Maverick. Our results provide useful information for the breeding of various soybean cultivars by plant biotechnology such as, genome editing.

• Journal of Life Science
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• v.29 no.6
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• pp.631-636
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• 2019

A new nitric oxide-induced (NOI) gene was isolated by screening ESTs from a cDNA library of dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). The 720 bp cDNA fragment, IbNOI, was sequenced, from which a 77 amino acid residue protein was deduced. A search of the protein BLAST database identified significant similarity to other plant NOI protein sequences. Quantitative RT-PCR analysis revealed diverse expression patterns of IbNOI in various tissues of the intact sweetpotato plant, and in leaves exposed to different stresses. The IbNOI gene was highly expressed in storage roots and suspension-cultured cells. In leaf tissues, IbNOI showed strong expression during sodium nitroprusside (SNP)-induced NO accumulation and chemical stress treatments. Expression of IbNOI was also induced under various abiotic stress conditions, such as dehydration, salt, and bacterial pathogen infection. These results suggest that IbNOI is involved in plant responses to diverse abiotic stresses and pathogen infection through a NO-related pathway.

• Journal of Practical Agriculture & Fisheries Research
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• v.23 no.1
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• pp.117-128
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• 2021

The skeletal muscle development of Hanwoo steer has been processed in the prenatal and postnatal periods. Bovine satellite cell located in perimysium of muscle tissues has differentially distributed in peripheral tissues. The study of postnatal development of satellite cells can help understand the genetic and functional regulation of meat characteristics. Factors affecting muscle size increase are related to the accumulation of DNA or synthesis of RNA proteins. In this study, we observed muscle development and differentiation after culturing bovine satellite cells derived from longissimus dorsi and semimembranosus regions of Hanwoo muscle tissue. In addition, RNA sequencing data were analyzed for differentially expressed genes (DEG) involved in intracellular muscle development and growth. The DEG of the two muscle tissues were compared according to 1day, 2day, 4day, and 7day. The overall gene expression level was confirmed by the heat map. Gene Ontology (GO) classification method was used to compare the expression level of gene groups affecting LD and SM development. The histology of GO was consistent with the time-cause change of LD and SM cell morphology. SM showed more active skeletal muscle development than LD. Even within the same time, SM expressed more genes than LD, thus synthesizing more muscle fibers

• Animal Bioscience
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• v.36 no.4
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• pp.654-670
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• 2023

Objective: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples. Methods: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method. Results: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63℃ for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×10² CFU/mL (6 CFU/reaction) and 9.01 pg/μL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×10⁴ CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method. Conclusion: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.

• KSBB Journal
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• v.23 no.6
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• pp.519-524
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• 2008

Monacolin-K is a strong anti-hypercholesterolemic agent produced by Monascus sp. via polyketide pathway. High-yielding mutants of monacolin-K were developed through rational screening strategies adopted based on understanding of monacolin-K biosynthetic pathway. Through the experiments for investigating various amino acids as putative precursors for the monacolin-K biosynthesis, it was found that production level of monacolin-K was remarkably increased when optimum amount of cysteine was supplemented into the production medium. We suggested that these phenomena might be related to the special roles of SAM (S-adenosyl methionine), a putative methyl group donor in the biosynthetic pathway of monacolin-K, demonstrating close interrelationship between SAM-synthesizing primary metabolism and monacolin-K synthesizing secondary metabolism. Namely, increase in the intracellular amount of SAM derived from the putative precursor, cysteine which was extracellularly supplemented into the production medium might contribute to the significant enhancement in the monacolin-K biosynthetic capability of the highly mutated producers. On the basis of these assumptions derived from the above fermentation results, we decided to construct efficient expression vectors harboring SAM synthetase gene (metK) cloned from A. nidulans, with the hope that increased intracellular level of SAM could lead to further enhancement in the monacolin-K production through overcoming a rate-limiting step associated with monacolin-K biosynthesis. Hence, in order to overcome the plausible rate-limiting step associated with monacolin-K biosynthesis by increasing intracellular level of SAM, we transformed the producer mutants with an efficient expression vector harboring gpdA promoter of the producer microorganism, and metK gene. Notably, from the resulting various transformants, we were able to screen a very high-yielding transformant which showed approximately 3.3 fold higher monacolin-K productivity than the parallel nontransformed mutants in shake flask cultures performed under the identical fermentation conditions.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.112-118
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• 2002

Sporulation in the fission yeast Schizosaccharomyces pombe has been regarded as an important model of cellular development and differentiation. S. pombe cells proliferate by mitosis and binary fission on growth medium. Deprivation of nutrients especially nitrogen sources, causes the cessation of mitosis and initiates sexual reproduction by matting between two sexually compatible cell types. Meiosis is then followed in a diploid cell in the absence of nitrogen source. DNA fragment complemented with the mutations of sporulation gene was isolated from the S. pombe gene library constructed in the vector, pDB 248' and designated as pDB(spo5)1. We futher analyzed six recombinant plasmids, pDB(spo5)2, pDB(spo5)3, pDB(spo5)4, pDB(spo5)5, pDB (spo5)6, pDB(spo5)7 and found each of these plasmids is able to rescue the spo5-2, spo5-3, spo5-4, spo5-5, spo5-6, spo5-7 mutations, respectively. Mapping of the integrated plasmid into the homologous site of the S. pombe chromosomes demonstrated that pDB(spo5)1, and pDB(spu5)Rl contained the spo5 gene. Transcripts of spo5 gene were analyzed by Northern hybridization. Two transcripts of 3.2 kb and 2.5kb were detected with 5kb Hind Ⅲ fragment containing a part of the spo5 gene as a probe. The small mRNA(2.5kb) appeared only when a wild-type strain was cultured in the absence of nitrogen source in which condition the large mRNA (3.2kb) was produced constitutively. Appearance of a 2.5kb spo5-mRNA depends upon the function of the meil, mei2 and mei3 genes.

• Clinical and Experimental Pediatrics
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• v.48 no.2
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• pp.197-203
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• 2005

Purpose : Changes in metalloproteinases(MMP) activity have been demonstrated in several disease states, including rheumatoid arthritis and tumor metastasis. More importantly, increased myocardial MMP activity has been reported to occur in both clinical and experimental forms of dilated cardiomyopathy. There was no report about MMP in adriamycin(ADR)-induced cardiomyopathy. The purpose of this study was to investigate gene expression of MMP and tissue inhibitor of metalloproteinases(TIMP) in ADR-induced cardiomyopathy and clarify the relationship between MMP and cytokines. Methods : Male Sprague-Dawley rats were divided into two groups. The first group was control. The second group was given intraperitoneal injections of ADR(5 mg/kg) twice a week over two weeks. Serum concentrations of MMP, TIMP, interleukin(IL)-6 and tumor necrosis factor(TNF)-${\alpha}$ were measured. RNA extraction was performed from frozen rat hearts. Reverse transcription polymerase chain reaction(RT-PCR) was employed. cDNA Microarray analysis was performed by using a set of 5,184 sequence-verified rat cDNA clones. Results : Serum MMP and TIMP levels were not significantly different between the two groups. IL-6 was $36.8{\pm}2.8pg/mL$ and TNF-${\alpha}$ $2.2{\pm}2.7pg/mL$ in the ADR group. They were significantly higher than in the control group. Serum MMP correlated significantly with TNF-${\alpha}$(r=0.41, P<0.05). There was no gene expression of MMP, IL-6 or TNF-${\alpha}$ in the hearts of both groups. Gene expression of TIMP was significantly depressed in the hearts of the ADR group. Conclusion : These results suggested a potential role for TNF-${\alpha}$ in the regulation of extracellular matrix remodeling in ADR induced cardiomyopathy. Rapid screening of multiple decreased gene expression by DNA chip may be a useful diagnostic test to detect early cardiac injury before developing ADR induced cardiomyopathy.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.15 no.1
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• pp.1-8
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• 2015

Purpose: 3-methylcrotonyl CoA carboxylase deficiency (3MCCD) is leucine metabolic disorder caused by mutation in MCCC1 or MCCC2 gene. Clinical manifestations are variable, ranging from fatal neonatal onset to asymptomatic individuals. There is no retrospective study of Korean patients undergoing long-term treatment for 3MCCD. We reported this study to find out clinical symptoms and gene analysis of 3MCCD patients. Methods: This study was based on data of patients diagnosed with 3MCCD in Soonchunhyang university hospital between April 2009 and September 2013. We report clinical, enzymatic and mutation data of 3MCCD patients found by newborn screening. Results: In tandem mass spectrometry, 3-OH-isovalerylcarnitine (C5OH) of all patients increased. And all 7 patients were elevated 3-methylcrotonylglycine (3MCG) and 3-hydroxyisovaleric acid (3HIVA) in urine. MCCC mutation was identified in 2 patients and MCCC2 was mutated in 5 patients. We found mutation occurred in 8 different parts of nucleotide and such mutation caused 7 different types of changes in amino acid. All patients are on medication of L-carnitine and L-glycine. 4 patients are taking biotin. And 4 patients are eating leucine free formula. After starting treatment, there were no significant changes of urine 3MCG and 3HIVA levels. Conclusions: According to our data, MCCC2 gene mutation was more common than MCCC1 gene mutation. But the level of 3HIVA or 3MCG in urine has no correlation with phenotype. All patients has no symptoms and are shown normal development.