• Title, Summary, Keyword: target gene

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Dual-Target Gene Silencing by Using Long, Synthetic siRNA Duplexes without Triggering Antiviral Responses

  • Chang, Chan Il;Kang, Hye Suk;Ban, Changill;Kim, Soyoun;Lee, Dong-ki
    • Molecules and Cells
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    • v.27 no.6
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    • pp.689-695
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    • 2009
  • Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of non-specific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without non-specific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.

Effects of different target sites on antisense RNA-mediated regulation of gene expression

  • Park, Hongmarn;Yoon, Yeongseong;Suk, Shinae;Lee, Ji Young;Lee, Younghoon
    • BMB Reports
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    • v.47 no.11
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    • pp.619-624
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    • 2014
  • Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability.

Genomic Analysis of miR-21-3p and Expression Pattern with Target Gene in Olive Flounder

  • Jo, Ara;Lee, Hee-Eun;Kim, Heui-Soo
    • Genomics & Informatics
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    • v.15 no.3
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    • pp.98-107
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    • 2017
  • MicroRNAs (miRNAs) act as regulators of gene expression by binding to the 3' untranslated region (UTR) of target genes. They perform important biological functions in the various species. Among many miRNAs, miR-21-3p is known to serve vital functions in development and apoptosis in olive flounder. Using genomic and bioinformatic tools, evolutionary conservation of miR-21-3p was examined in various species, and expression pattern was analyzed in olive flounder. Conserved sequences (5'-CAGUCG-3') in numerous species were detected through the stem-loop structure of miR-21-3p. Thus, we analyzed target genes of miR-21-3p. Among them, 3' UTR region of PPIL2 gene indicated the highest binding affinity with miR-21-3p based on the minimum free energy value. The PPIL2 gene showed high expression levels in testis tissue of the olive flounder, whereas miR-21-3p showed rather ubiquitous expression patterns except in testis tissue, indicating that miR-21-3p seems to control the PPIL2 gene expression in a complementary repression manner in various tissues of olive flounder. Taken together, this current study contributes to infer the target gene candidates for the miR-21-3p using bioinformatics tools. Furthermore, our data offers important information on the relationship between miR-21-3p and target gene for further functional study.

Multiple shRNA expressing vector enhances efficiency of gene silencing

  • Song, Jun;Giang, An;Lu, Yingchun;Pang, Shen;Chiu, Robert
    • BMB Reports
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    • v.41 no.5
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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.

Grp78 is a Novel Downstream Target Gene of Hoxc8 Homeoprotein

  • Kang, Jin-Joo;Bok, Jin-Woong;Kim, Myoung-Hee
    • Biomedical Science Letters
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    • v.17 no.1
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    • pp.1-5
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    • 2011
  • Previously, we have identified 14 putative downstream target genes of Hoxc8 homeoprotein in F9 murine embryonic teratocarcinoma cells through proteomics analysis. Among those, we tested a possibility of a DNA-k type molecular chaperone, Grp78, as a direct downstream target of Hoxc8, by cloning a 2.4 kb upstream region of murine Grp78 into a reporter plasmid and by testing if Hoxc8 can regulate its expression. We observed that Hoxc8 proteins could transactivate the reporter gene, which was affected by small interference RNAs (siRNAs) against to Hoxc8, suggesting that Grp78 is a novel downstream target of Hoxc8 in vivo.

Prediction of Hypoxia-inducible Factor Binding Site in Whale Genome and Analysis of Target Genes Regulated by Predicted Sites (고래의 게놈에서 hypoxia-inducible factor binding site의 예측과 target gene에 대한 분석)

  • Yim, Hyung-Soon;Lee, Jae-Hak
    • Journal of Marine Bioscience and Biotechnology
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    • v.7 no.2
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    • pp.35-41
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    • 2015
  • Whales are marine mammals that are fully adapted to aquatic environment. Whales breathe by lungs so they require adaptive system to low oxygen concentration (hypoxia) while deep and prolonged diving. However, the study for the molecular mechanism underlying cetacean adaptation to hypoxia has been limited. Hypoxia-inducible factor (HIF) is the central transcription factor that regulates hypoxia-related gene expression. Here we identified HIF-binding sites in whale genome by phylogenetic footprinting and analyzed HIF-target genes to understand how whales cope with hypoxia. By comparison with the HIF-target genes of terrestrial mammals, it was suggested that whales may retain unique adaptation mechanisms to hypoxia.

Efficient Production of loxP Knock-in Mouse using CRISPR/Cas9 System

  • Jung, Sundo
    • Biomedical Science Letters
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    • v.26 no.2
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    • pp.114-119
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    • 2020
  • Of the various types of mice used for genome editing, conditional knock-out (cKO) mice serve as an important model for studying the function of genes. cKO mice can be produced using loxP knock-in (KI) mice in which loxP sequences (34 bp) are inserted on both sides of a specific region in the target gene. These mice can be used as KO mice that do not express a gene at a desired time or under a desired condition by cross-breeding with various Cre Tg mice. Genome editing has been recently made easy by the use of third-generation gene scissors, the CRISPR-Cas9 system. However, very few laboratories can produce mice for genome editing. Here we present a more efficient method for producing loxP KI mice. This method involves the use of an HDR vector as the target vector and ssODN as the donor DNA in order to induce homologous recombination for producing loxP KI mice. On injecting 20 ng/µL of ssODN, it was observed that the target exon was deleted or loxP was inserted on only one side. However, on injecting 10 ng/µL of the target HDR vector, the insertion of loxP was observed on both sides of the target region. In the first PCR, seven mice were identified to be loxP KI mice. The accuracy of their gene sequences was confirmed through Sanger sequencing. It is expected that the loxP KI mice produced in this study will serve as an important tool for identifying the function of the target gene.

Insight into Norfloxacin Resistance of Acinetobacter oleivorans DR1: Target Gene Mutation, Persister, and RNA-Seq Analyses

  • Kim, Jisun;Noh, Jaemin;Park, Woojun
    • Journal of Microbiology and Biotechnology
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    • v.23 no.9
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    • pp.1293-1303
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    • 2013
  • Antibiotic resistance of soilborne Acinetobacter species has been poorly explored. In this study, norfloxacin resistance of a soil bacterium, Acinetobacter oleivorans DR1, was investigated. The frequencies of mutant appearance of all tested non-pathogenic Acinetobacter strains were lower than those of pathogenic strains under minimum inhibitory concentration (MIC). When the quinolone-resistance-determining region of the gyrA gene was examined, only one mutant (His78Asn) out of 10 resistant variants had a mutation. Whole transcriptome analysis using a RNA-Seq demonstrated that genes involved in SOS response and DNA repair were significantly up-regulated by norfloxacin. Determining the MICs of survival cells after norfloxacin treatment confirmed some of those cells were indeed persister cells. Ten colonies, randomly selected from among those that survived in the presence of norfloxacin, did not exhibit increased MIC. Thus, both the low mutation frequency of the target gene and SOS response under norfloxacin suggested that persister formation might contribute to the resistance of DR1 against norfloxacin. The persister frequency increased without a change in MIC when stationary phase cells, low growth rates conditions, and growth-deficient dnaJ mutant were used. Taken together, our comprehensive approach, which included mutational analysis of the target gene, persister formation assays, and RNA sequencing, indicated that DR1 survival when exposed to norfloxacin is related not only to target gene mutation but also to persister formation, possibly through up-regulation of the SOS response and DNA repair genes.

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila

  • Kim, Keon-Hee;Yoo, Siuk
    • BMB Reports
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    • v.47 no.2
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    • pp.92-97
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    • 2014
  • We have examined the effect of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulated the castor gene expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5'-TTAT-3' canonical core motifs than the probe containing the 5'-TTAC-3' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes.

Selective Detection of Campylobacter jejuni, C. coli, Arcobacter butzleri and Helicobacter pylori by Polymerase Chain Reaction (Campylobacter jejuni, C. coli, Arcobacter butzleri와 Helicobacter pylori의 PCR에 의한 분리검출)

  • Lee, Young-Duck;Park, Jong-Hyun
    • Korean Journal of Food Science and Technology
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    • v.34 no.6
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    • pp.1134-1139
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    • 2002
  • Campylobacter, Arcobacter, and Helicobacter, classified into the same rRNA superfamily VI by taxonomy, cause food-borne diseases, stomach ulcer, and gastric cancer. To detect each strain selectively from contaminated foods, PCR, multiplex-PCR, and restricion fragment length polymorphism (RFLP) were applied on Campylobacter, Arcobacter, and Helicobacter. The same PCR products could be detected using CHA primer targeted for 16S rRNA of Campylobacter, Arcobacter, and Helicobacter. To detect C. jejuni and C. coli from A. butzleri and H. pylori, pg50/pg3 primer targeted for fla A gene was used, and for A. butzleri, Arco2/Butz primer targeted for 23S rRNA was utilized. For H. pylori detection, icd1/icd2 primer targeted for isocitrate dehydrogenase gene was employed, and JEJ1/JEJ2 primer targeted for ceuE gene was effective for C. jejuni detection from the three strains. C. jejuni, C. coli could be separated from A. butzleri and H. pylori through PCR-RFLP using restriction enzyme Dde I. Such primers would be effective for detecting each strain selectively through PCR when C. jejuni, C. coli, A butzleri and H. pylori are contaminated together.