• Proceedings of the PSK Conference
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• 2003.10a
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• pp.60-61
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• 2003

Human Genome Project has recently been completed and the information on nucleotide sequences of our whole genome is now available at the public or commercial data banks. Next goals are to identify the functions of each gene and to elucidate the intracellular signal transduction pathways regulating gene expression. We have established a PCR-based bioassay to search for biologically active compounds that can modulate the expression of genes encoding important proteins. (omitted)

• IMMUNE NETWORK
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• v.2 no.4
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• pp.233-241
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• 2002

Background: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. Methods: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain ${\lambda}$ genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. Results: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. Conclusion: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.7
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• pp.4209-4214
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• 2013

Background: Breast cancer is among the five most common cancers and ranks first among cancers diagnosed in Iranian women. Screening and treatment of this disease with molecular methods, especially regarding high incidences at early age and advanced stage, is essential. Several genes with altered expression have been identified by cDNA microarray studies in breast cancer, with the Bcl-2 gene indicated as a likely candidate. In this study, we studied Bcl-2 gene expression levels in parallel tumor and non-tumor breast tissues. Materials and Methods: Forty samples including 21 tumor, 16 non tumor (marginal) and 3 benign breast tissues which were all pathologically diagnosed, were subjected to RNA extraction and polyA RT-PCR with the expression level of Bcl-2 quantified using real-time PCR. Results: There is higher expression levels of the Bcl-2 gene in tumor samples compared with marginal samples, but not attaining significance(p>0.05). Bcl-2 expression in 14 (66.7%) of the cases of tumor samples and 9 (56.3%) cases of the marginal samples were positive. Comparison of the expression of the Bcl-2 gene in histological grade showed that a high expression of Bcl-2 was associated with a high histological grade (p<0.41). Conclusions: Our data suggests that dysregulated Bcl-2 gene expression is potentially involved in the pathogenesis of breast cancer. Using gene expression analysis may significantly improve our ability for screening cancer patients and will prove a powerful tool in the diagnosis and prognostic evaluation of the disease whilst aiding the cooperative group trials in the Bcl-2 based therapy project.

• Horticultural Science & Technology
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• v.29 no.4
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• pp.345-351
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• 2011

This study was carried out to develop a model system using selection method for disease resistant plant breeding programs using a herbicide bialaphos-resistant patII gene as a gene-based marker. Spraying bialaphos could eliminate the susceptible plants from the segregating populations such as ${F_2}^{\prime}s$ and thereafter. Tomato cv. Momotaro-yoke was transformed with patII gene 60 independent transformants were acquired. Total 42 transformants were analyzed in transgene copy numbers by Southern blotting and the segregation ratios for the bialaphos resistance. Statistical analysis revealed that the transgene copy numbers and the segregation ratios were not always coincided, especially having the tendency of underestimating the real numbers of the transgenes in the multicopy lines. A two-stepwise screening method was applied to select $T_1$ tomato plants which linked the transgenic patII to a disease resistance gene (I2 and Ve). Based on the resistant to susceptible ratios, T-20 plant was finally selected due to the estimated linkage 12-13 cM between the patII gene to the I2 gene on chromosome 11. This newly developed system could be applied to any economical crop in breeding programs.

• 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 Life Science
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• v.16 no.2 s.75
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• pp.360-364
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• 2006

Fibrin clots of blood vessels are one of the serious factor caused cardiovascular disease. The development of a antithrombotic and thrombolysis solvent is necessary to prevent and treat these diseases. It has been reported that a strong fibrin-specific fibrinolytic enzyme was produced from a Korean fermented soybean paste similar to Japanese miso. We have been screened the known or novel fibrinolytic enzymes by activity-based and sequence-based screening from soil DNA metagenome library containing all kinds of environmental genomic DNA. The activity-based screening was determined the protease activity on 0.5% skim milk. For sequence-based screening, we designed a set of primer expanding gene sequence of fibrinolytic enzyme, performed PCR and selected clones showing the expected size of amplicons from metagenome library. Transformation of the gene encoding fibrinolytic enzyme was carried out with commercial vectors and their transformants were selected. Finally, we found 15 positive clones from metagenome library. Then each of sequences were analyzed and identified as similar or known the clones of nattokinase. We are going to perform full sequence of each clones, ligate with expression vector, transform into competent cells and then determine activity of expressed enzymes.

• Childhood Kidney Diseases
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• v.24 no.2
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• pp.131-137
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• 2020

Nephrogenic diabetes insipidus (DI) is a rare disease in which the patient cannot concentrate urine despite appropriate or high secretion of antidiuretic hormone. Congenital nephrogenic DI is caused by the arginine vasopressin receptor 2 (AVPR2) or aquaporin 2 (AQP2) gene mutation; the AVPR2 genetic mutation accounts for 90% of the cases. National health screening for infants and children was launched in 2007 in order to prevent accidents and promote public health in infants and children in Korea. The program has been widely used as a primary clinical service in Korea. We treated an infant with faltering growth and delayed development detected by the National health screening program, and diagnosed the problem as nephrogenic DI caused by a rare missense mutation of c.490T>C on the AVPR2 gene. This case can be a good educational nephrogenic DI with a rare AVPR2 mutation, which was well screened and traced by the national health screening program for infants and children in Korea.

• Genomics & Informatics
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• v.14 no.4
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• pp.160-165
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• 2016

Over the past decade, the detection of gene-gene interactions has become more and more popular in the field of genome-wide association studies (GWASs). The goal of the GWAS is to identify genetic susceptibility to complex diseases by assaying and analyzing hundreds of thousands of single-nucleotide polymorphisms. However, such tests are computationally demanding and methodologically challenging. Recently, a simple but powerful method, named "BOolean Operation-based Screening and Testing" (BOOST), was proposed for genome-wide gene-gene interaction analyses. BOOST was designed with a Boolean representation of genotype data and is approximately equivalent to the log-linear model. It is extremely fast, and genome-wide gene-gene interaction analyses can be completed within a few hours. However, BOOST can not adjust for covariate effects, and its type-1 error control is not correct. Thus, we considered two-step approaches for gene-gene interaction analyses. First, we selected gene-gene interactions with BOOST and applied logistic regression with covariate adjustments to select gene-gene interactions. We applied the two-step approach to type 2 diabetes (T2D) in the Korea Association Resource (KARE) cohort and identified some promising pairs of single-nucleotide polymorphisms associated with T2D.

• Animal cells and systems
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• v.5 no.1
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• pp.65-69
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• 2001

Obtaining mutant animals is important for studying the function of a particular gene. A chemical mutagenesis was first carried out to generate mutations in C. elegans. In this study, we used ultraviolet-activated 4,5',8-trimethylpsoralen to induce small deletion mutations. A library of mutagenized worms was prepared for recovery of candidate animals and stored at $15^{\circ}C$ during screening instead of being made into a frozen stock library. In order to isolate deletion mutations in target genes, a polymerase chain reaction (PCR)-based screening method was used. As a result, two independent mutants with deletions of approximately 1.0 kb and 1.3 kb were isolated. This modified and improved reverse genetic approach was proven to be effective and practical for isolating mutant animals to study gene function at the organismal level.

• 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.