• BMB Reports
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• v.28 no.6
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• pp.538-545
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• 1995

When DNA replication of simian virus 40 (SV40) is initiated on the replication origin, the regions containing the initiation sites of DNA primase, which participates in the transient RNA primer synthesis for formation of Okazaki fragments in the lagging strand, were chosen as the target sites of antisense RNA for studies of the inhibition of SV40 DNA replication. Four recombinant transcription vectors, pUC-PrI, pUC-PrII, pGEM-PrBS, and pGEM-PrSN, coding antisense RNA, were constructed. Four antisense RNAs (named as I, II, BS, and SN) having the size of 18, 19,58, and 123 nts, respectively, were made from the transcription vectors by in vitro transcription. And then, antisense RNA in the concentration of 2${\mu}m$ were added to COS cells transfected with pATSV-W which is a recombinant plasmid containing the SV40 origin of replication. The inhibitory extent of DNA replication was measured by DpnI resistance and was confirmed by measurement of transient RNA primer synthesis. The result shows that six combinations of antisense RNA (I, II, BS, SN, I+SN, and BS+SN) lead to the inhibition of SV40 DNA replication by up to 85%.

• Biomedical Science Letters
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• v.10 no.2
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• pp.65-73
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• 2004

I report that single-stranded antisense as a part of large circular (LC-) genomic DNA of recombinant M13 phage exhibits enhanced stability, sequence specific antisense activity, and no need for target site search. A cDNA fragment (708 bp) of rat TNF-$\alpha$ was inserted into a phagemid vector, and TNF-$\alpha$ antisense molecules (TNF$\alpha$-LCAS) were produced as single-stranded circular DNA. When introduced into a rat monocyte/macrophage cell line, WRT7/P2, TNF$\alpha$-LCAS was able to ablate LPS-induced TNF-$\alpha$ mRNA to completion. The antisense effect of TNF$\alpha$-LCAS was shown to be sequence-specific because expressions of three control genes ($\beta$-actin, GAPDH and IL-1$\beta$) were not significantly altered by the antisense treatment. Further, TNF$\alpha$-LCAS was found to be highly efficacious as only 0.1 $\mu$g (0.24 nM) of TNF$\alpha$-LCAS was sufficient to block TNF-$\alpha$ expression in 1$\times10^5$ WRT7/P2 cells. I have also observed specific antisense activity in reduction of NF-$\kappa$B gene expression. The results suggest that an antisense sequence as a part of single-stranded circular genomic DNA has a specific antisense activity.

• Journal of Photoscience
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• v.9 no.2
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• pp.451-453
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• 2002

A new concept of "photo" -antisense method has been evaluated, where the inhibition of gene expression by the conventional antisense method is enhanced by photochemical binding between antisense oligonucleotides conjugated with photo-reactive compound and target mRNA or DNA. Fluorescein labeled oligodeoxyribonucleotides (F-DNA) was delivered to cell nuclei in the encapsulated form in multilamellar lecithin liposomes with neutral charge. F-DNA was previously shown to photo-bind to the complementary stranded DNA, and the delivery system using neutral liposome to be effective in normal human keratinocytes. In the present study, we used human kidney cancer G401.2/6TG.1 cell line to be advantageous in reproducible experiments. p53 was adopted as a target gene since antisense sequence information has been accumulated. The nuclear localization ofF-DNA was identified by comparing the fluorescence ofF-DNA with that of Hoechst 33258 under fluorescence microscope. After 7hr incubation to accumulate p53 protein induced by UV -B, p53 protein was quantified by Western blot. After 2hrs from F-DNA application, about 30% of cell population incorporated F-DNA in their nuclei with some morphological change possibly due to liposomal toxicity. Irradiation of visible light longer than 400nm from solar simulator at this time enhanced the inhibitory action of antisense F-DNA. The present results suggest that photo-antisense method is promising to control gene expression in time and space dependent manner. Further improvement of F-DNA delivery to cancer cells in the stability and toxicity is in progress. progress.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.131-134
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• 1998

$\textrm{T}_{5}$ progeny of one transgenic tomato line (To9) carrying antisense polygalacturonase (PG) cDNA was generated by selfing. Five $\textrm{T}_{5}$ plants were used to analyse in detail. The PG antisense gene was stably inherited through fifth generations. In all five $\textrm{T}_{5}$ plants, expression of the antisense transcripts were detected. In consequence, it led to a reduction of the PG enzyme activity in ripe fruit to between 37% and 65% that of normal. In two plants the expression of endogenous PG gene was inhibited in ripe fruit.

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.103-108
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• 2000

We have cloned and analyzed cDNA coding for non-virion (NV) protein of the m V - P R T The NV gene contained 336 bp open readmg frame and encoded a protein of 11 1 amino acids with a molecular weight of 13.2 kDa. The deduced amino acid sequence of NV of IHNVPRT was found to be 90-95% identical to those of foreign isolates of IHNV. These results indicate that NV gene of the MNV is highly conserved among &ifferent strains of THNV Northern blot analyses revealed that the levels of NV gene expression were strongly elevated after 20 h post-infection. In order to identify the role of NV in the replication of MNV in fish cells, IHNVinfected cells were treated with antisense oligonucleotides. While IHNV-PRT exposed to glycoprotein (G) antisense oligonucleotide showed severely reduced growth, the growth of virus exposed to NV antisense oligonucleotide was not affected by NV antisense oligonucleotide, which suggests that NV is not essential for replication of IHNV in fish cells.

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.351-355
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• 1995

A truncated Polygalacturonase (PG) cDNA was fused in reverse orientation to the CaMV 35S promoter of the binary vector pCA643, and introduced into tomato cells by Agrobaderium - mediated transformation. Transformed cells were selected using kanamycin as select agent then regenerated into plants. After selfed, one transgenic line (T9), was germinated and grown on MS medium containing 1 mg/mL of kanamycin Genomic Southern analysis of a T9 progeny with labelled PG2 cDNA probe showed a single antisense PC fragment as well as the endogenous PG2 gene, suggesting that PC antisense gene was integrated into tomato genome. Northern blot analysis demonstrated that the antisense RNA was produced from the transgene at much tiger level than the endogenous PG2 gene. Polygalacturonase activity analysis of the fruit from transgenic plants demonstrated that the antisense transgene expression caused 4 to 60% reduction of endogenous PG activity.

• Microbiology and Biotechnology Letters
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• v.38 no.1
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• pp.70-76
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• 2010

The single-stranded large circular (LC)-sense DNA were utilized as probes for DNA chip experiments. The microarray experiment using LC-sense DNA probes found differentially expressed genes in A549 cells as compared to WI38VA13 cells, and microarray data were well-correlated with data acquired from quantitative real-time RT-PCR. A 5K LC-sense DNA microarray was prepared, and the repeated experiments and dye swap test showed consistent expression patterns. Subsequent functional analysis using LC-antisense library of overexpressed genes identified several genes involved in A549 cell growth. These experiments demonstrated proper feature of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense microarray and antisense libraries for an effective functional validation of genes.

• Journal of Plant Biotechnology
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• v.40 no.4
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• pp.192-197
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• 2013

This study examined the phenotypic and molecular characteristics of the $2^{nd}$ clone ($T_0V_2$) plants of LeLs-antisense gene-transgenic chrysanthemum line (LeLs80) that exhibited non-branching, proving the relevance of these characteristics as a factor for use in environmental risk assessment. Results of the Southern blot analysis showed that three copies of the LeLs-antisense gene were introduced into the transgenic line, and northern analysis showed that the transcripted gene was normally expressed in the transgenic line. A flanking T-DNA sequencing method was used to determine that sequences of 184 and 464 bps flanked the LeLs-antisense gene in the transgenic line. These sequences, respectively, matched the 35S promoter for expression of the npt II gene and the NOS terminator for expression of the LeLs-antisense gene within the pCAMBIA 2300 vector.

• BMB Reports
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• v.28 no.3
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• pp.243-248
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• 1995

Cholesteryl ester transfer protein (CETP), a hydrophobic glycoprotein promoting transfer of cholesteryl esters (CE) from high-density lipoproteins (HDL) to lower-density lipoproteins in the plasma, has been recognized a potent atherogenic factor during the development of coronary artery diseases. This study demonstrated a possible utilization of antisense RNA to inhibit expression of the CETP gene using vaccinia virus as an expression system. The CETP cDNA was inserted into a transfer vector (pSC11) in sense and antisense orientations and used to generate recombinant viruses. Recombinants containing sense or antisense orientations of the CETP cDNA were isolated by $TK^-$ selection and X-gal test. The inserted CETP cDNAs in the recombinants were identified by Southern blot analysis and allowed to transcribe in host cells (CV-1). Expressions of the exogenous CETP mRNA, extracted from the CV-1 cells coinfected with viruses containing sense and antisense DNAs, were monitored by Northern blot analysis using the CETP cDNA probe, by Western blot analysis using monoclonal antibody against the C-terminal active region of the CETP and by the CETP assay. Decreased expressions of the exogenous CETP cDNA were clearly evident in the Northern and Western blot analyses as the dose of antisense expression increased. In the CETP assay, the CETP activities decreased compared to the activity obtained from the cell extracts infected with sense construct only.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.29 no.2
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• pp.86-94
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• 2003

It has been known that transglutaminase C (TGase C, TGase II) is directly participated in the DNA organization of chromosome, and affects the cellular processes such as proliferation, differentiation, and apoptosis of cells, but still not known what mechanism is working on. In this study, the cytogenetic and the immunohistochemical methods were used to observe the TGase C expression in the nuclear chromosome of the proliferating cells, especially in mitotic stage. The human gastric adenocarcinoma (SNU-1) cell line was used for immunohistochemistry and antisense inhibition study in vitro. The present study was also aimed to disclose the efficiency of antisense inhibition by using antisense oligonucleotide DNA labeled with fluorescence, and found that anti-TGase C probe was diffusely infiltrated into the cytoplasm and the nucleus of the cell. By the antisense inhibition the nuclei of SNU-1 cells became rough nuclear shape, as they were greatly reduced in TGase C immunoreactivity both for the normal and apoptotic SNU-1 cells. However, it is clearly presumed that the TGase C directly interacts with the chromosome of SNU-1 cells and it may play an important role in the division and organization of the chromosome during the mitotic stage.