• 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.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.29-29
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• 2003

Incompatible plant-pathogen interactions result in the rapid cell death response known as hypersensitive response (HR) and activation of host defense-related genes. To understand the molecular and cellular mechanism controlling defense response better, several approaches including isolation and characterization of novel genes, promoter analysis of those genes, protein-protein interaction analysis and reverse genetic approach etc. By using the yeast two-hybrid system a clone named Tsipl, Tsil -interacting protein 1, was isolated whose translation product apparently interacted with Tsil, an EREBP/AP2 type DNA binding protein. RNA gel blot analysis showed that the expression of Tsipl was increased by treatment with NaCl, ethylene, salicylic acid, or gibberellic acid. Transient expression analysis using a Tsipl::smGFP fusion gene in Arabidopsis protoplasts indicated that the Tsipl protein was targeted to the outer surface of chloroplasts. The targeted Tsipl::smGFP proteins were diffused to the cytoplasm of protoplasts in the presence of salicylic acid (SA) The PEG-mediated co-transfection analysis showed that Tsipl could interact with Tsil in the nucleus. These results suggest that Tsipl-Tsil interaction might serve to regulate defense-related gene expression. Basically the useful promoters are valuable tools for effective control of gene expression related to various developmental and environmental condition.(중략)

• Korean Journal of Biological Psychiatry
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• v.4 no.1
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• pp.48-53
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• 1997

CV(bDAT) cell line, expressing dopamine transporter stably, has been established by transfection of CV-1 cells with bovine dopamine transporter cDNA. Using CV(bDAT) cells, the effects of various antipsychotic drugs on dopamine uptake activity were investigated. All of antipsychotic drugs tested, inhibited the [$^3H$]dopamine uptake into CV(bDAT) cells with $IC_{50}s$ in the low to mid micromolar range, implying that antipsychotic drugs may produce overflow of dopamine in the synaptic cleft of dopaminergic neuron.

• Polymer(Korea)
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• v.34 no.6
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• pp.501-506
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• 2010

Natural chitosan has high molecular weight and the poor solubility in water. Water-soluble chitosan with low molecular weight was prepared by the hydrolysis method. In order to develop an efficient gene delivery carrier, chitosan was conjugated with lipoic acid to form the comb-type copolymer. The copolymer with the amphiphilic property formed the self-assembled nanoparticles in the aqueous solution. The average size of nanoparticles was 217.6 nm and the average size of nanoparticles/DNA complex was 170 nm. New chitosan-lipoic acid copolymer showed the low cytotoxicity and 10 times higher transfection efficiency than that of the pure chitosan.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.329-334
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• 2006

Cationic liposome has been studied as one of the most promising non-viral gene delivery systems. In this report, we describe a new synthesized cholesterolic cationic lipid (2-aminoethylcarbamate-cholesterol) and dioleylphosphatidylethanolamine (DOPE) improve the cellular uptake properties of antisense ODNs, as well as plasmid DNA with low toxicity. This formulation of cholesterolic cationic lipid termed Chol-E, efficiently transfects ODNs and plasmids into many cell types in the presense or absence of 10% serum in the medium.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.6
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• pp.483-489
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• 2009

Despite the potential importance of the human regulator of calcineurin 1 (RCAN-1) gene in the modulation of cell survival under stress, little is known about its role in death-inducing signal pathways. In this study, we addressed the effects of RCAN1.4 knockdown on cellular susceptibility to apoptosis and the activation of death pathway proteins. Transfection of siRNAs against RCAN1.4 resulted in enhanced Fas- and etoposide-induced apoptosis, which was associated with increased expression and translocation of Bax to mitochondria. Our results suggest that enhanced expression and activation of p53 was responsible for the upregulation of Bax and the increased sensitivity to apoptosis, which could be reversed by p53 knockdown. To explain the observed upregulation of p53, we propose a downregulation of the ubiquitin ligase HDM2, probably translationally. These findings show the importance of appropriate RCAN1.4 expression in the modulation of cell survival and reveal a link between RCAN1.4 and p53.

• Journal of Korean Biological Nursing Science
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• v.7 no.1
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• pp.47-56
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• 2005

nm23-H1 gene expression has been inversely correlated with tumor metastatic potential in certain tumors including melanomas, breast carcinomas, and hepatocellular carcinomas. However, its role with respect to the invasive behavior of central nervous system tumors has scarcely been addressed Because cell motility and invasion plays an essential role in metastatic dissemination, we have studied whether motile human glioma cell(U87MG) transfected with nm23-H1 complementary DNA have any alterations in their ability to migrate and invade. There was no significant changes in the shape and size of the cells following nm23-H1 transfection. The role of nm23-H1 in glioma migration and invasion have been evaluated by in vitro simple scratch technique and brain slice invasion model Basal migration ability of nm23-H1 transfectants cell(U87MG-pEGFP-nm23) were lesser than U87MG. Accordingly, U87MG-pEGFP-nm23 didn't migrate away apparently from the tumors implanted site comparing U87MG in brain slice invasion model. These results suggest that nm23-H1 may play an important role in suppressing the human glioma migration and invasion.

• BMB Reports
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• v.30 no.4
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• pp.269-273
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• 1997

Heptocvte-specific expression induced by Hepatitis B virus (HBV) enhancer 2-core gene promoter was examined in various hepatocyte and non-hepatocyte cell lines. using non-viral and retroviral vector systems in which chloramphenicol acetyltransferase (CAT) is used as a reporter. The non-viral plasmid containing the HBV enhancer 2-core promoter exhibited 22 and 66% of CAT activities in hepatoma cell lines. HepG2 and Hep3B, respectively when compared with CAT activity expressed by CMV promoter. The CAT activities, however. were found to be marginal in other tested hepatoma cell lines as well as mouse primary hepatocytes and non-hepatocytes. The HBV enhancer 2 located upstream the CMV promoter did not affect the CMV promoter activity nor provided hepatocyte-specific expression. Transfection of retroviral plasmid DNA containing the HBV enhancer 2-core promoter as an internal promoter exhibited high and specific CAT expression in HepG2 and Hep3B cell lines but the activity value was 5 to 10 fold lower than the non-viral plasmid with identical promoter. These results suggest that the usage of HBV enhancer 2-core promoter for liver specific expression is limited to certain vectors and hepatocyte cell lines.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2000.04a
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• pp.17-19
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• 2000

Heterotrimeric G Proteins transduce ligand binding to a wide variety of seven transmembrane cell surface receptors into intracellular signals. The currently accepted model for the activation of G protein suggests that ligand-activated receptor accelerates GDP-GTP exchange reactions on the ${\alpha}$ subunit of the heterotrimeric G protein. At least seventeen distinct isoforms of the G${\alpha}$ subunit protein have been identified in mammalian organisms. Among them, the G${\alpha}$q family consists of five members whose ${\alpha}$ subunits show different expression patterns. G${\alpha}$q and G${\alpha}$11 seem to be almost ubiquitously expressed, whereas G${\alpha}$14 is predominantly expressed in spleen, lung, kidney and testis. G${\alpha}$16 and its murine counterpart G${\alpha}$15 are expressed in hematopoietic cells and has been shown to couple a wide variety of receptors to phosphoinositide-specific phospholipase C activity. Beta-isoforms of phospholipase C were shown to be activated by all members of G${\alpha}$q family, i.e., G${\alpha}$q, G${\alpha}$11, G${\alpha}$l4 and G${\alpha}$16 subunits either in reconstitution system. or in experiments using cDNA transfection with intact Cos-7 cells.

• BMB Reports
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• v.41 no.2
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• pp.146-152
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• 2008

In the presence of NGF, PC12 cells extend neuronal processes, cease cell division, become electrically excitable, and undergo several biochemical changes that are detectable in developing sympathetic neurons. We investigated the expression pattern of the apoptosis-related genes at each stage of neuronal differentiation using a cDNA microarray containing 320 apoptosis-related rat genes. By comparing the expression patterns through time-series analysis, we identified candidate genes that appear to regulate neuronal differentiation. Among the candidate genes, HO2 was selected by real-time PCR and Western blot analysis. To identify the roles of selected genes in the stages of neuronal differentiation, transfection of HO2 siRNA in PC12 cells was performed. Down-regulation of HO2 expression causes a reduction in neuronal differentiation in PC12 cells. Our results suggest that the HO2 gene could be related to the regulation of neuronal differentiation levels.