• Fisheries and Aquatic Sciences
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• 제13권1호
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• pp.56-62
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• 2010

To determine whether long double-stranded RNA (dsRNA) induces RNA interference and type I interferon (IFN) responses in fish, long dsRNAs encoding enhanced green fluorescent protein (EGFP), GFPuv, and polyinosinic-polycytidylic acid sequences were co-injected with an EGFP expressing plasmid, into rock bream (Oplegnathus fasciatus). We investigated the EGFP mRNA and protein levels, and the transcriptional responses of dsRNA-dependent protein kinase and Mx1 genes. Long dsRNAs were strong inducers of a type I IFN response in rock bream, resulting in nonspecific suppression of exogenous gene expression. Furthermore, sequence-specific knockdown of exogenous gene expression at the mRNA level was detected at an early phase (24 h). These results suggested that long dsRNA may inhibit exogenous gene expression through an early mRNA interference response and a later type I IFN response in fish.

• 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.

• Animal cells and systems
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• 제11권2호
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• pp.99-104
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• 2007

RNA interference (RNAi) is the phenomenon of gene silencing by double-stranded RNA (dsRNA) at transcriptional and post-transcriptional levels in a sequence-specific manner. Reverse genetic approaches using RNA interference (RNAi) have become a major tool for biological researches since its discovery in the nematode Caenorhabditis elegans. In this review, we overview how the RNAi phenomenon was discovered and how the underlying mechanism has been elucidated. We also describe and discuss how RNAi experiments can be performed and how RNAi can be used for genetic studies.

• Asian Pacific Journal of Cancer Prevention
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• 제16권16호
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• pp.7205-7210
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• 2015

Background: In this study, influence caused by expression plasmids of connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) short hairpin RNA (shRNA) on mRNA expression of CTGF,TIMP-1,procol-${\alpha}1$ and PCIII in hepatic tissue with hepatic fibrosis, a precancerous condition, in rats is analyzed. Materials and Methods: To screen and construct shRNA expression plasimid which effectively interferes RNA targets of CTGF and TIMP-1 in rats. 50 cleaning Wistar male rats are allocated randomly at 5 different groups after precancerous fibrosis models and then injection of shRNA expression plasimids. Plasmid psiRNA-GFP-Com (CTGF and TIMP-1 included), psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA-DUO-GFPzeo of blank plasmid are injected at group A, B, C and D, respectively, and as model control group that none plasimid is injected at group E. In 2 weeks after last injection, to hepatic tissue at different groups, protein expression of CTGF, TIMP-1, procol-${\alpha}1$ and PC III is tested by immunohistochemical method and,mRNA expression of CTGF,TIMP-1,procol-${\alpha}1$ and PCIII is measured by real-time PCR. One-way ANOVA is used to comparison between-groups. Results: Compared with model group, there is no obvious difference of mRNA expression among CTGF,TIMP-1,procol-${\alpha}1$, PC III and of protein expression among CTGF, TIMP-1, procol-${\alpha}1$, PC III in hepatic tissue at group injected with blank plasmid. Expression quantity of mRNA of CTGF, TIMP-1, procol-${\alpha}1$ and PCIII at group A, B and C decreases, protein expression of CTGF, TIMP-1, procol-${\alpha}1$, PC III in hepatic tissue is lower, where the inhibition of combination RNA interference group (group A) on procol-${\alpha}1$ mRNA transcription and procol-${\alpha}1$ protein expression is superior to that of single interference group (group B and C) (P<0.01 or P<0.05). Conclusions: RNA interference on CTGF and/or TIMP-1 is obviously a inhibiting factor for mRNA and protein expression of CTGF, TIMP-1, procol-${\alpha}1$ and PCIII. Combination RNA interference on genes of CTGF and TIMP-1 is superior to that of single RNA interference, and this could be a contribution for prevention of precancerous condition.

• BMB Reports
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• 제43권4호
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• pp.291-296
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• 2010

Cyclin-dependent kinase 2 (CDK2) is a member of serine/threonine protein kinases, which initiates the principal transitions of the eukaryotic cell cycle and is a promising target for cancer therapy. The present study was designed to inhibit cdk2 gene expression to induce cell cycle arrest and cell proliferation suppression. Here, we constructed a series of RNA interference (RNAi) plasmids which can successfully express small interference RNA (siRNA) in the transfected human cells. The results showed that the RNAi plasmids containing the coding sequences for siRNAs down-regulated the cdk2 gene expression in human cancer cells at the mRNA and the protein levels. Furthermore, we found that the cell cycle was arrested at G0G1 phases and the cell proliferation was inhibited by different siRNAs. These results demonstrate that suppression of CDK2 activity by RNAi may be an effective strategy for gene therapy in human cancers.

• Fisheries and Aquatic Sciences
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• 제17권3호
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• pp.377-380
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• 2014

RNA interference (RNAi)-mediated transcriptional knock-down of Crassostrea gigas big defensin 1 and 2 genes (Cg-BigDef1 and Cg-BigDef2) was investigated. The cDNA sequences of Cg-BigDef1 and Cg-BigDef2 were identical, excluding an additional fragment of 20 nucleotides in Cg-BigDef1; thus, a long double-stranded RNA (dsRNA) targeting the mRNA of Cg-BigDef2 effectively downregulated both Cg-BigDef2 and Cg-BigDef1. In addition, long dsRNA targeting green fluorescent protein (GFP) did not affect transcription of the two big defensin genes. These results suggest that the transcriptional downregulation of Cg-BigDef1 and Cg-BigDef2 was mediated by sequence-specific RNA interference (RNAi). Despite injection of long dsRNA targeting Cg-BigDef2 into only the adductor muscle, knock-down of Cg-BigDef1 and Cg-BigDef2 was observed in the adductor muscle, hemocytes, mantle, and gills, suggestive of systemic spread of RNAi in C. gigas. Furthermore, the inhibitory effect of dsRNA persisted until 72 h post-injection, indicative of a long-lasting RNAi-mediated knock-down of target genes.

• Clinical and Experimental Reproductive Medicine
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• 제30권4호
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• pp.299-307
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• 2003

Objectives: The present study was conducted to evaluate the mobile transposon-like element, clone MTi7 (MTi7) expression in the mouse ovary and to determine its role(s) in the mouse oocytes by RNA interference (RNAi). Methods: MTi7 mRNA expression was localized by in situ hybridization in day5 and adult ovaries. Double stranded RNA (dsRNA) was prepared for c-mos, a gene with known function as control, and the MTi7. Each dsRNA was microinjected into the germinal vesicle (GV) stage oocytes then oocyte maturation and intracellular changes were evaluated. Results: In situ hybridization analysis revealed that MTi7 mRNA localized to the oocyte cytoplasm from primordial to preovulatory follicles. After dsRNA injection, we found 43-54% GV arrest of microinjected GV oocytes with 68%-90% decrease in targeted c-mos or MTi7 mRNA. Conclusions: This is the first report of the oocyte-specific expression of the MTi7 mRNA. From results of RNAi for MTi7, we concluded that the MTi7 is involved in the germinal vesicle breakdown in GV oocytes, and MTi7 may be implicated with c-mos for its function. We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions.

• 한국응용곤충학회지
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• 제56권2호
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• pp.153-164
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• 2017

이중나선형 RNA (dsRNA)를 이용한 유전자 발현 억제기술이 다양한 생명체에서 기능 유전체학을 연구하는 데 이용되고 있다. 이 기술의 원리는 전사후 단계에서 유전자 발현을 조절하는 RNA 간섭에 기인된다. dsRNA를 이용하여 특정 유전자의 발현 억제는 심각한 치사효과를 줄 수 있다. 이러한 분자기작을 해충 방제에 적용하여 특정 dsRNA를 이용한 새로운 살충제를 개발하고 있다. 본 종설은 dsRNA를 이용한 RNA간섭 원리를 설명하고 이를 이용한 해충 방제를 구현한 여러 예를 살펴본다. 그리고 해충방제를 실현시키기 위해 현재 이 기술이 담고 있는 한계를 고찰하고 이에 대한 대응 방안을 본 종설에서 제공하고자 한다.

• BMB Reports
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• 제41권5호
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• pp.358-362
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• 2008

RNA interference (RNAi) is the process of sequence-specific gene silencing. However, RNAi efficiency still needs to be improved for effective inhibition of target genes. We have developed an effective strategy to express multiple shRNAs (small hairpin RNA) simultaneously using multiple RNA Polymerase III (Pol III) promoters in a single vector. Our data demonstrate that multiple shRNAs expressed from Pol III promoters have a synergistic effect in repressing the target gene. Silencing of endogenous cyclophilin A (CypA) or key HIV viral genes by multiple shRNAs results in significant inhibition of the target gene.

• 한국발생생물학회지:발생과생식
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• 제12권2호
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• pp.159-168
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• 2008

Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain the undifferentiated state of stem cells. However, molecular mechanisms underlying the function of Nanog remain largely unknown. To elucidate the regulatory roles of Nanog involved in maintenance of P19 embryonal carcinoma (EC) stem cells, we transfected three small interfering RNA (siRNA) duplexes targeted against different regions of the Nanog gene into P19 cells. The Nanog siRNA-100 duplexes effectively decreased the expression of Nanog up to 30.7% compared to other two Nanog siRNAs, the Nanog siRNA-400 (67.9 %) and -793 (53.0%). When examined by RT-PCR and real-time PCR, the expression of markers for pluripotency such as Fgf4, Oct3/4, Rex1, Sox1 and Yes was downregulated at 48 h after transfection with Nanog siRNA-100. Furthermore, expression of the ectodermal markers, Fgf5 and Isl1 was reduced by Nanog knockdown. By contrast, the expression of other markers for pluripotency such as Cripto, Sox2 and Zfp57 was not affected by Nanog knockdown at this time. On the other hand, the expression of Lif/Stat3 pathway molecules and of the endoderm markers including Dab2, Gata4, Gata6 and the germ cell nuclear factor was not changed by Nanog knockdown. The results of this study demonstrated that the knockdown of Nanog expression by RNA interference in P19 cells was sufficient to modulate the expression of pluripotent markers involved in the self-renewal of EC stem cells. These results provide the valuable information on potential downstream targets of Nanog and add to our understanding of the function of Nanog in P19 EC stem cells.