• Molecules and Cells
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• v.27 no.5
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• pp.577-582
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• 2009

The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon ${\gamma}$, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells.

• Applied Biological Chemistry
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• v.39 no.4
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• pp.260-264
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• 1996

Process of particle bombardment for efficient transformation of Cymbidium virescence rhizome microcross sections was investigated using Biolistic particle delivery system with pBI121 harboring the ${\beta}-glucuronidase$(GUS) and the neomycin phosphotransferaseII(nptII). The best result was obtained from the combination of $1.11{\;}{\mu}m$ tungsten particles coated with pBl121, $77.33kg/cm^2$ helium pressure, 6.35 mm gap distance, and 7.0 cm target distance. Transient expression of the reporter gene, GUS, bombarded into the rhizome microsections was observed by the histochemical assay. The marker gene, nptII, delivered by bombarding the tungsten particles coated with the plasmid DNA was identified in the transformed rhizome by polymerase chain reaction.

• Journal of Life Science
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• v.9 no.1
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• pp.15-21
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• 1999

The random amplified ploymorphic DNAs (RAPD) assay is a simple and useful tool in identification of appropriate genetic markers, that requires no knowledge of target DNA sequence. RAPD products were generated directly from seaweed tissues, without prior nucleic acid extraction, of Porphyra yezoensis, Ulva pertusa and Undaria pinnatifida. The nuclear rDNA internal transcribed spacer (ITS) fragment however was not amplified directly from the seaweed tissues. Using DNA extracted by the LiCl method, both the ITS and RAPD's have been amplified by the polymerase chain reaction. RAPD of P yezoensis, thallus (n) and conchocelis (2n) produced lots of different polymorphic bands (36-50$\%$) depending on the arbitrary primers used. Difference was also observed between direct tissues amplification and DNA extracts amplification (53-57$\%$). Thus it is important to use the same ploidy of tissue for DNA extraction and as a RAPD template.

• Korean Journal of Environmental Agriculture
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• v.40 no.3
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• pp.186-193
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• 2021

BACKGROUND: Before generating transgenic plant using the CRISPR-Cas9 system, the efficiency test of sgRNAs is recommended to reduce the time and effort for plant transformation and regeneration process. The efficiency of the sgRNA can be measured through the transient expression of sgRNA and Cas9 gene in tomato cotyledon; however, we found that the calculated efficiency showed a large variation. It is necessary to increase the precision of the experiment to obtain reliable sgRNA efficiency data from transient expression. METHODS AND RESULTS: The cotyledon of 11^th, 15^th, 19^th, and 23^rd-day-old tomato (Solanum lycopersicum cv. Micro-Tom) were used for expressing CRISPR-Cas9 transiently. The agrobacterium harboring sgRNA for targeting ALS2 gene of tomato was injected through the stomata of leaf adaxial side and the genomic DNA was extracted in 5 days after injection. The target gene edition was identified by amplifying DNA fragment of target region and analyzing with Illumina sequencing method. The target gene editing efficiency was calculated by counting base deletion and insertion events from total target sequence read. CONCLUSION: The CRISPR-Cas9 editing efficiency varied with tomato cotyledon age. The highest efficiency was observed at the 19-day-old cotyledons. Both the median and mean were the highest at this stage and the sample variability was also minimized. We found that the transgene of CRISPR-Cas9 system was strongly correlated with plant leaf development and suggested the optimum cotyledon leaf age for Agrobacterium-mediated transfection in tomato.

• Development and Reproduction
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• v.16 no.4
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• pp.379-384
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• 2012

Lhx8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian folliculogenesis. However, Lhx8 DNA binding sequences are not characterized yet. We aimed to identify and characterize a cis-acting sequence of germ-cell specific transcriptional factor, Lhx8. To identify Lhx8 DNA binding element, Cyclic Amplification of Sequence Target (CAST) Analysis was performed. Electrophoretic Mobility Shift Assay (EMSA) was processed for the binding specificity of Lhx8. Luciferase assay was for the transcriptional activity of Lhx8 through identified DNA binding site. We identified a putative cis-acting sequence, TGATTG as Lhx8 DNA binding element (LBE). In addition, Lhx8 binds to the LBE with high affinity and augments transcriptional activity of luciferase reporter driven by artificial promoter containing the Lhx8 binding element. These findings indicate that Lhx8 directly regulates the transcription of genes containing Lhx8 binding element in oocytes during early folliculogenesis.

• Journal of Microbiology
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• v.44 no.1
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• pp.42-49
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• 2006

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

• Proceedings of the Korean Information Science Society Conference
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• 2003.04c
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• pp.455-457
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• 2003

DNA microarray는 분자생물학에서 널리 사용되고 있는 실험 도구로써 크게 cDNA와 oligonucleotide microarray로 나뉘어진다. DNA microarray는 일련의 DNA 서열로 이루어진 probe들의 집합으로 구성되며 알려지지 않은 서열과의 hybridization 과정을 통해 특정 서열을 인식할 수 있게 된다. O1igonucieotide microarray는 cDNA 방법과는 다르게 probe를 구성하는 서열을 제작자가 임의로 구성할 수 있기 때문에 목표 서열이 가지는 고유한 부분만을 probe 서열로 사용함으로써 비용절감과 실험의 정확도를 높일 수 있다는 장점이 있다. 그러나 현재 목표 유전자 서열에 대해 probe 집합을 생성하는 결정적인 방법은 존재하지 않으며, 따라서 넓은 해 공간에서 효과적으로 최적 해를 찾아 주는 진화 연산이 probe 선택을 위한 좋은 대안으로 사용될 수 있다［1.2］. 그러나 진화연산을 이용한 probe 선택방법에 있어서 인식하고자 하는 목표 서열의 개수가 많아질 경우, 해 공간의 크기가 커짐으로 인해 문제점이 발생할 수 있다. 따라서 본 논문에서는 다수의 목표 유전자 서열을 대상으로 한 probe 선택 방법에 일어서 보다 효율적인 진화연산 접근 방법을 소개한다. 제시된 방법은 인식하고자 하는 목표 서얼의 일부를 선택해 이를 probe 집합의 후보로 사용하며. 유전 연산자를 이용한 진화과정을 통해 최적에 가까운 probe 집합을 찾는다. 본 논문은 GenBank로부터 유전자 서열을 대상으로 제안된 방법을 실험하였으며, 축소된 목표 서열만을 이용해 probe 집합을 선택하더라도 적합한 probe 집합을 찾을 수 있었다.

• BMB Reports
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• v.52 no.2
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• pp.151-156
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• 2019

RAD51 recombinase plays a critical role in homologous recombination and DNA damage repair. Here we showed that expression of RAD51 is frequently upregulated in lung cancer tumors compared with normal tissues and is associated with poor survival (hazard ratio (HR) = 2, P = 0.0009). Systematic investigation of lung cancer cell lines revealed higher expression of RAD51 in KRAS mutant (MT) cells compared to wildtype (WT) cells. We further showed that MT KRAS, but not WT KRAS, played a critical role in RAD51 overexpression via MYC. Moreover, our results revealed that KRAS MT cells are highly dependent on RAD51 for survival and depletion of RAD51 resulted in enhanced DNA double strand breaks, defective colony formation and cell death. Together, our results suggest that mutant KRAS promotes RAD51 expression to enhance DNA damage repair and lung cancer cell survival, suggesting that RAD51 may be an effective therapeutic target to overcome chemo/radioresistance in KRAS mutant cancers.

• Genomics & Informatics
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• v.3 no.3
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• pp.53-62
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• 2005

MicroRNAs play an important role in regulating gene expression, but their target identification is a difficult task due to their short length and imperfect complementarity. Burge and coworkers developed a program called TargetScan that allowed imperfect complementarity and established a procedure favoring targets with multiple binding sites conserved in multiple organisms. We improved their algorithm in two major aspects - (i) using well-defined UTR (untranslated region) database, (ii) examining the extent of conservation inside the 3' UTR specifically. Average length in our UTR database, based on the ECgene annotation, is more than twice longer than the Ensembl. Then, TargetScan was used to identify putative binding sites. The extent of conservation varies significantly inside the 3' UTR. We used the 'tight' tracks in the UCSC genome browser to select the conserved binding sites in multiple species. By combining the longer 3' UTR data, TargetScan, and tightly conserved blocks of genomic DNA, we identified 107 putative target genes with multiple binding sites conserved in multiple species, of which 85 putative targets are novel.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.2
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• pp.125-132
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• 2006

Objective: In the previous study, we complied the differentially expressed genes during early folliculogenesis. Objective of the present study was to identify downstream target genes of transcription factors (TFs) using bioinformatics for selecting the target TFs among the gene lists for further functional analysis. Materials & Methods: By using bioinformatics tools, constituent domains were identified from database searches using Gene Ontology, MGI, and Entrez Gene. Downstream target proteins/genes of each TF were identified from database searches using TF database ($TRANSFAC^{(R)}$ 6.0) and eukaryotic promoter database (EPD). Results: DNA binding and trans-activation domains of all TFs listed previously were identified, and the list of downstream target proteins/genes was obtained from searches of TF database and promoter database. Based on the known function of identified downstream genes and the domains, 3 (HNF4, PPARg, and TBX2) out of 26 TFs were selected for further functional analysis. The genes of wee1-like protein kinase and p21WAF1 (cdk inhibitor) were identified as potential downstream target genes of HNF4 and TBX2, respectively. PPARg, through protein-protein interaction with other protein partners, acts as a transcription regulator of genes of EGFR, p21WAF1, cycD1, p53, and VEGF. Among the selected 3 TFs, further study is in progress for HNF4 and TBX2, since wee1-like protein kinase and cdk inhibitor may involved in regulating maturation promoting factor (MPF) activity during early folliculogenesis. Conclusions: Approach used in the present study, in silico analysis of downstream target genes, was useful for analyzing list of TFs obtained from high-throughput cDNA microarray study. To verify its binding and functions of the selected TFs in early folliculogenesis, EMSA and further relevant characterizations are under investigation.