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Shin, Mi-Ra,Cui, Xiang-Shum,Jun, Jin-Hunh,Jeong, Yu-Jeong,Kim, Nam-Hyung Wiley Subscription Services, Inc., A Wiley Company 2005 Molecular reproduction and development Vol.70 No.4
<P>Oct-4 is an essential transcription factor involved in the differentiation of the inner cell mass (ICM) in mouse blastocysts, and is thought to be the pluripotent gene of embryonic stem cells. However, downstream genes of Oct-4 and the mechanism by which it regulates the transcription machinery remain unclear. Here, we specifically knocked down Oct-4 gene expression in mouse blastocysts by double-stranded RNA (dsRNA) interference. A recently developed method, the annealing control primer (ACP) technique, was then used to identify the downstream genes of Oct-4. By using 120 arbitrary ACP, 10 clones were found to be differentially expressed in the knocked down embryos and the cloned genes were analyzed by DNA sequencing and BLAST searching. Quantitative real time reverse transcription (RT)-polymerase chain reaction (PCR) confirmed that the expression of these genes is altered by Oct-4 knockdown. Of the 10 genes, 8 (Atp6ap2, GK003, Ddb1, hRscp, Dppa1, Dpp3, Sap18, and Rent1) were downregulated and 2 (Rps14 and ETIF2B) were upregulated in Oct-4 dsRNA-injected blastocysts. One of the downregulated genes is developmental pluripotency associated-1 (Dppa1), which has already been identified as being an Oct-4 downstream gene. Two other genes, Rent1 and Sap18, were found to be Oct-4 downstream genes for the first time. The genes identified here will provide insights into the roles played by Oct-4 during embryonic development. Mol. Reprod. Dev. 70: 390–396, 2005. © 2005 Wiley-Liss, Inc.</P>
Gene Knockdown in the Olfactory Bulb, Amygdala, and Hypothalamus by Intranasal siRNA Administration
Il-Doo Kim,Seung-Woo Kim,Ja-Kyeong Lee 대한해부학회 2009 Anatomy & Cell Biology Vol.42 No.4
Intranasal administration provides a method of bypassing the blood brain barrier, which separates the systemic circulating system and central interstitial fluid, and directly drugs to the central nervous system. This method also circumvents first-pass elimination by the liver and gastrointestinal tract. In the present study, the authors investigated intranasal siRNA delivery efficiency by using FITC-labeled transfection control siRNA and a genespecific siRNA. The localization of fluorescence-tagged siRNA revealed that siRNA was delivered to cells in the olfactory bulb and that the level of the siRNA target gene(alpha B-crystallin) was significantly reduced in the same area. siRNA was delivered to processes as well as nuclei and cytoplasm. At 12 hrs after intranasal delivery, siRNA-mediated target gene reduction was observed in other more distally located brain regions, for example, in the amygdala, entorhinal cortex, and hypothalamus. Target gene knockdown was demonstrated by double immunohistochemistry, which demonstrated target gene suppression was detected not only in neurons but in glia, for example, astrocytes. These results indicate that intranasal siRNA delivery offers an efficient means of reducing specific target genes in certain regions of the brain and of performing gene knockdown-mediated therapy.
Gene Knockdown in the Olfactory Bulb, Amygdala, and Hypothalamus by Intranasal siRNA Administration
김일두,김승우,이자경 대한해부학회 2009 Anatomy & Cell Biology Vol.42 No.4
Intranasal administration provides a method of bypassing the blood brain barrier, which separates the systemic circulating system and central interstitial fluid, and directly delivering drugs to the central nervous system. This method also circumvents first-pass elimination by the liver and gastrointestinal tract. In the present study, the authors investigated intranasal siRNA delivery efficiency by using FITC-labeled transfection control siRNA and a genespecific siRNA. The localization of fluorescence-tagged siRNA revealed that siRNA was delivered to cells in the olfactory bulb and that the level of the siRNA target gene (alpha B-crystallin) was significantly reduced in the same area. siRNA was delivered to processes as well as nuclei and cytoplasm. At 12 hrs after intranasal delivery, siRNA-mediated target gene reduction was observed in other more distally located brain regions, for example, in the amygdala, entorhinal cortex, and hypothalamus. Target gene knockdown was demonstrated by double immunohistochemistry, which demonstrated alpha B crystallin expression depletion in more than 70% of cells at 12 hrs after the intranasal delivery. siRNA-mediated target gene suppression was detected not only in neurons but in glia, for example, astrocytes. These results indicate that intranasal siRNA delivery offers an efficient means of reducing specific target genes in certain regions of the brain and of performing gene knockdown-mediated therapy.
Gene Expression Knockdown by Modulating Synthetic Small RNA Expression in Escherichia coli
Noh, M.,Yoo, S.M.,Kim, W.J.,Lee, S.Y. Cell Press 2017 Cell systems Vol.5 No.4
<P>Escherichia coli gene expression knockdown using synthetic small RNA (sRNA) can be fine-tuned by altering sRNA sequences to modulate target mRNA-binding ability, but this requires thorough checking for off-target effects. Here, we present an sRNA gene expression knockdown system fine-tuned by using different promoters to modulate synthetic sRNA abundance. Our approach entails selecting knockdown target genes resulting from in silico flux response analysis and those related to product biosynthesis then screening strains transformed with a library of synthetic sRNA-promoter combinations for enhanced production. We engineered two E. coli strains, both utilizing fine-tuned repression of argF and glnA through our approach; one produced putrescine (42.3 +/- 1.0 g/L) and the other L-proline (33.8 +/- 1.6 g/L) by fed-batch culture. Fine-tuned gene knockdown by controlling sRNA abundance will be useful for rapid design of microbial strains through simultaneously optimizing expression of multiple genes at a systems level, as it overcomes the difficulties of constructing and testing many different sRNAs and checking their cross-reactivity.</P>
Silencing the cleavage factor CFIm25 as a new strategy to control Entamoeba histolytica parasite
Juan David Ospina-Villa,Nancy Guillén,Ce´sar Lo´pez-Camarillo,Jacqueline Soto-Sanchez,Esther Ramirez-Moreno,Raul Garcia-Vazquez,Carlos A. Castañon-Sanchez,Abigail Betanzos,Laurence A.Marchat 한국미생물학회 2017 The journal of microbiology Vol.55 No.10
The 25 kDa subunit of the Clevage Factor Im (CFIm25) is an essential factor for messenger RNA polyadenylation in human cells. Therefore, here we investigated whether the homologous protein of Entamoeba histolytica, the protozoan responsible for human amoebiasis, might be considered as a biochemical target for parasite control. Trophozoites were cultured with bacterial double-stranded RNA molecules targeting the EhCFIm25 gene, and inhibition of mRNA and protein expression was confirmed by RT-PCR and Western blot assays, respectively. EhCFIm25 silencing was associated with a significant acceleration of cell proliferation and cell death. Moreover, trophozoites appeared as larger and multinucleated cells. These morphological changes were accompanied by a reduced mobility, and erythrophagocytosis was significantly diminished. Lastly, the knockdown of EhCFIm25 affected the poly(A) site selection in two reporter genes and revealed that EhCFIm25 stimulates the utilization of downstream poly(A) sites in E. histolytica mRNA. Overall, our data confirm that targeting the polyadenylation process represents an interesting strategy for controlling parasites, including E. histolytica. To our best knowledge, the present study is the first to have revealed the relevance of the cleavage factor CFIm25 as a biochemical target in parasites.
전현수,이종범 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0
RNA interference(RNAi) technology can be applied for the treatment of malignant tumor cells, because it can target specific cells and induce apoptosis while minimizing direct effects on tumor specific protein expressing mRNA level.(e.g. siRNA) However, delivering siRNA to target cells problem due to the large reactivity of the RNA is still present. It introduces a particleization method with using a polymer with siRNA to solve the siRNA stability problems in SERUM condition, but there is no standardized considering cytotoxicity or interfering with the action of siRNA. In this study, we introduced a method of using calcium ion as a surface coater for RNA complex. Calcium is already abundant in the body for osteogenesis, neurochemical response et al. so there is no need to consider toxicity in vivo. Coating the RNA complex to determine the likelihood of a calcium ion, and then we analyzed for morphological changes of the complexes and to perform in vitro cell experiment on Hela cells.