• Journal of Plant Biotechnology
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• 제34권4호
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• pp.307-312
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• 2007

클라미도모나스 (Chlamydomonas reinhardtii)에서 Tombus 바이러스의 p19 유전자가 RNAi suppressor로서 기능하는 지는 조사하기 위하여 형질전환을 수행하였다. Southern분석을 통하여 p19 유전자가 1 copy에서 여러 copy로 도입된 형질전환체를 선발하였다. MAA7 유전자의 3'UTR 부위의 inverted repeat (IR) DNA가 형질전환되어 제작된 #33 strain을 p19유전자로 다시 형질전환 한 결과, MAA7 mRNA의 발현 양이 변하여 일정하지 않고 교란되었으며, 5-FI가 첨가된 배지에서 증식이 저해되었다. 증식이 저해된 것은 p19 유전자에 의한 silencing의 suppression이 일어난 것으로 간주할 수 있다. 그러나 MAA7 mRNA의 발현양이 전반적으로 증가하지 않고 발현양의 교란이 일어났으므로 p19 유전자에 의한 silencing의 suppression으로 보기 어렵다. 따라서 본 연구에 사용된 p19 유전자가 클라미도모나스에서는 codon usage가 상이하여 단백질 발현양이 충분치 않아서 silencing의 suppression을 완전하게 하지 못하거나 고등식물에서 일어나는 방식과는 다른 방식으로 suppression한다고 할 수 있다.

• The Plant Pathology Journal
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• 제28권1호
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• pp.81-86
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• 2012

We developed a reassortant RNA virus vector derived from $Cucumber$ $mosaic$ $virus$ (CMV), which has advantages of very wide host range and can efficiently induce gene silencing in a few model plants. Certain CMV isolates, however, show limited host ranges presumably because they naturally co-evolved with their own hosts. We used a reassortant comprised of two strains of CMV, Y-CMV and Gn-CMV, to broaden the host range and to develop a virus vector for virus-induced gene silencing (VIGS). Gn-CMV could infect chili pepper and tomato more efficiently than Y-CMV. Gn-CMV RNA1, 3 and Y-CMV RNA2-A1 vector were newly reconstructed, and the transcript mixture of RNA1 and 3 genomes of Gn-CMV and RNA2 genome of Y-CMV RNA2 containing portions of the endogenous phytoene desaturase (PDS) gene (CMV2A1::PDSs) was inoculated onto chili pepper (cv. Chung-yang), tomato (cvs. Bloody butcher, Tigerella, Silvery fir tree, and Czech bush) and $Nicotiana$ $benthamiana$. All the tested plants infected by the reassortant CMV vector showed typical photo-bleaching phenotypes and reduced expression levels of $PDS$ mRNA. These results suggest that the reassortant CMV vector would be a useful tool for the rapid induction of the RNA silencing of endogenous genes in chili pepper and tomato plants.

• Asian Pacific Journal of Cancer Prevention
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• 제13권3호
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• pp.1043-1048
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• 2012

The COPS3 gene has stimulating effect on cell proliferation and progression of osteosarcomas and related cells. However, the features of COPS3 and its potential application as a therapeutic target in other cancers has not yet been studied. In this study, therefore, the effect of COPS3 silencing via COPS3 siRNA on lung cancer cell proliferation was examined. Expression levels of COPS3 gene in COPS3 siRNA infected cells and control siRNA infected cells were compared with real time PCR and Western blot analysis. Cell proliferation levels were comprehensively analyzed by MTT, BrdU incorporationy, and colony formation assays. For mechanistic assessment the effects of COPS3 silencing on cell cycle and apoptosis were analyzed using flow cytometry. Results showed that successful silencing of the COPS3 gene at both translational and transcriptional levels significantly reduced the proliferation and colony formation by lung cancer cells (p<0.01). Flow cytometry showed cell cycle arrest in the G0/G1 phase after COPS3 silencing, and more importantly, apoptosis was induced as a result of COPS3 knockdown, which negatively affected cell survival. Therefore, these results provide another piece of important evidence that the COPS3 gene expressed in lung cancer cells may play a critical role in stimulating proliferation. Down-regulation of COPS3 could significantly inhibit lung cancer cell growth, which was most likely mediated via induction of cell cycle arrest in G0/G1 phase and apoptosis.

• Plant Biotechnology Reports
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• 제2권3호
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• pp.199-206
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• 2008

We have developed an efficient system of assessing the ability of a gene silencing cassette to silence transcripts from recalcitrant or poorly studied plant species by using a model plant as a host for the gene of interest. Tobacco plants transgenic for Lachrymatory Factor Synthase (LFS) enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. In this work, we have demonstrated that the RNAi construct is a suitable candidate for the development of a non-lachrymatory onion. Our model plant RNAi system has wide-reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in the pharmacological, food and process industries.

• Molecules and Cells
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• 제21권1호
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• pp.153-160
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• 2006

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature ($15^{\circ}C$) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1573-1579
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• 2018

Transcriptional gene silencing is regulated by the chromatin structure, which is by various factors including histones. Saccharomyces cerevisiae contains transcriptionally silenced regions such as telomeric regions and hidden mating (HM) loci. The positively-charged amino acids on the histone H4 tail were reported to be critical for the telomeric silencing in yeast, by interacting with Dot1, a specific methyltransferase for the $79^{th}$ lysine on histone H3. However, Dot1 did not affect gene silencing within HM loci, but whether the positively-charged amino acids on the H4 tail affect HM silencing has not been defined. To elucidate the function of the H4 tail on HM silencing, we created several MATa-type yeast strains bearing the substitution of arginine with alanine or lysine on the histone H4 tail and checked the sensitivity of MATa-type yeast to alpha pheromone. The arginine point mutants substituted by alanine (R17A, R19A, and R23A) did not show sensitivity to alpha pheromone, but only two arginine mutants substituted by lysine (R17K and R19K) restored the sensitivity to alpha pheromone-like wild type. These data suggested that the basic property of arginine at $17^{th}$ and $19^{th}$ positions in the histone H4 tail is critical for maintaining HM silencing, but that of the $23^{rd}$ arginine is not. Our data implicated that the positive charge of two arginine residues on the histone H4 tail is required for HM silencing in a manner independent of Dot1.

• Plant Biotechnology Reports
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• 제5권3호
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• pp.273-281
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• 2011

Diverse epigenetic phenotypes are frequently found during research on transgenic plants. To understand the factors underlying such diversity, hundreds of independent 35S-GFP transgenic N. benthamiana plants were analyzed. The diverse GFP-expression phenotypes of the transgenic plants were classified into three major types based on the GFP expression patterns and their response to 35S-GFP agroinfiltration: steady-green, silenced and non-uniform phenotype. The non-uniform phenotype was further sub-divided into five minor phenotypes: variegated, red-dropped, on-silencing, partitioned and misty, according to the distribution of GFP expression on the leaves. Many of transgenic plants continuously generated diverse phenotypes over several generations despite the transgene identity. Such epigenetic GFP phenotyping was found to be the result of spontaneous transgene silencing mediated by either or both of post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). This finding was verified by the detection of 21- and 24-nt small interfering RNA (siRNA) molecules, and DNA methylation in the transgenic plants that showed repeated epigenetic variation. Agroinfiltration demonstrated that irregular distribution of GFP on a leaf was the result of erratic transgene silencing, and the technique also proved to be a rapid and effective method for selecting fully silenced plants within 3 days. Furthermore, two novel phenotypes described are potential materials for in-depth investigations into the genes and mechanisms responsible for spontaneous transgene silencing.

• 원예과학기술지
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• 제30권6호
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• pp.734-742
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• 2012

본 연구는 배추의 유전자 기능분석을 위한 RNAi 유전자 침묵 기법과 T-DNA 삽입 기법을 비교하기 위해 수행하였다. 두 종류의 형질전환 계통이 이용되었으며 BrSAMS-knockout(KO) 계통은 T-DNA 삽입으로 한 개의 Brassica rapa S-adenosylmethionine synthetase(BrSAMS) 유전자가 기능을 상실한 계통이었으며 BrSAMS-knockdown(KD) 계통은 RNAi 방법을 통해 BrSAMS 유전자들의 발현이 억제된 계통이었다. KO 계통과 KD 계통의 microarray 분석 결과에서는 SAMS 유전자와 관련된 sterol, 자당, homogalacturonan 생합성 및 glutaredoxin-related protein, serine/threonine protein kinase, 그리고 gibberellin-responsive protein 유전자들의 발현 수준이 뚜렷한 차이를 보여 주었다. 그러나 KO 계통의 유전자 발현 양상은 하나의 BrSAMS 유전자가 기능을 상실하였음에도 불구하고 대조 계통과 비교하여 RNAi기법을 적용한 KD 계통에 비해 큰 차이를 보여주지 못했다. 또한 직접적으로 SAMS 유전자와 관련된 폴리아민과 에틸렌 합성 유전자들의 발현 변화도 KD 계통에서 더 잘 나타났다. 본 연구에서 microarray 결과를 이용한 KO 계통의 BrSAMS 기능분석은 배추과식물의 게놈 triplication 발생으로 인하여 다수로 존재하는 SAMS 유전자들 때문에 명확한 결론을 얻을 수 없었다. 결론적으로 배추와 같은 배수체 작물의 유전자 기능 분석은 RNAi silencing에 의한 유전자 knock-down 기법이 T-DNA 삽입에 의한 knock-out 기법보다 더욱 효율적인 것으로 나타났다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권3호
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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.

• Asian Pacific Journal of Cancer Prevention
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• 제14권4호
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• pp.2295-2299
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• 2013

Background: To explore the role of caveolin-1(CAV-1) gene silencing on MAPK activation in lipopolysaccharide (LPS)-challenged human mammary epithelial cells. Methods: We established a MCF-10ACE of CAV-1 gene silencing from human mammary epithelial cell line MCF-10A by RNAi technology. DNA Microarray were used to detect the expression of inflammation-associated genes in MCF10ACE. Western blotting was used to examine the activation of MAPK in lipopolysaccharide(LPS)-challenged MCF-10A and MCF-10ACE. Moreover, immunofluorescence and Western bloting were performed to detect the co-localization of CAV-1 and toll-like receptor 4 (TLR4) in human mammary epithelial cells. Results: MCF-10ACE exhibited significant increases in inflammation-associated gene expression, especially IL-6 (~7-fold) and IL6R (~17-fold). In addition, LPS-induced p38 MAPK and JNK MAPK activation was significantly increased in MCF-10ACE. Furthermore, CAV-1 co-localized with TLR4 and appeared a negative correlation trend. Conclusion: CAV-1 gene silencing promotes MAPK activation via TLR4 signaling in human mammary epithelial cells response to LPS.