• Journal of Microbiology and Biotechnology
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• 제30권11호
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• pp.1777-1784
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• 2020

To increase the availability of microalgae as producers of valuable compounds, it is necessary to develop novel systems for gene expression regulation. Among the diverse expression systems available in microalgae, none are designed to induce expression by low temperature. In this study, we explored a cold-inducible system using the antifreeze protein (AFP) promoter from a polar diatom, Chaetoceros neogracile. A vector containing the CnAFP promoter (pCnAFP) was generated to regulate nuclear gene expression, and reporter genes (Gaussia luciferase (GLuc) and mVenus fluorescent protein (mVenus)) were successfully expressed in the model microalga, Chlamydomonas reinhardtii. In particular, under the control of pCnAFP, the expression of these genes was increased at low temperature, unlike pAR1, a promoter that is widely used for gene expression in C. reinhardtii. Promoter truncation assays showed that cold inducibility was still present even when pCnAFP was shortened to 600 bp, indicating the presence of a low-temperature response element between -600 and -477 bp. Our results show the availability of new heterologous gene expression systems with cold-inducible promoters and the possibility to find novel low-temperature response factors in microalgae. Through further improvement, this cold-inducible promoter could be used to develop more efficient expression tools.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• 대한의생명과학회지
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• 제13권3호
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• pp.207-212
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• 2007

The purpose of this preliminary study is to improve the efficiency of gene transfer of nonviral plasmid DNA by in vivo electroporation in ischemic hindlimb muscle, tibialis anterior. Hindlimb ischemic model was aseptically made by excision of left femoral artery. Each $50\;{\mu}g$ of pEGFP-C1 and pGL3-control in $100\;{\mu}l$ 0.9% NaCl was injected in tibialis anterior muscle. In vivo electroporation was applied on the same site with 10 mm-distance 2 needle array electrodes and ECM830. In 3 groups of normal rat with different electric field strength 0, 200 and 800 V/cm, the expression of pEGFP-C1 was comparatively evaluated. In 8 groups of normal rats, the expression of pGL3-control was evaluated in 0, 40, 50, 80, 100, 150, 200 and 300 V/cm of electric field strength. In 5 groups of ischemic models, the expression of pGL3-control was analyzed on 0, 4, 7, 10 and 14 days elapsed after making ischemic models. In 9 groups of ischemic rats, the expression of pGL3-control was analyzed in the electric field strength 0, 60, 70, 80, 100, 150, 200, 250 and 300 V/cm. GFP expressions in normal tibialis anterior were high in the extent and degree in order of electric field strength of 200, 800 and 0 V/cm. Luciferase value was highest in $50{\sim}100\;V/cm$ electric field strength. In the case of ischemic models, luciferase expression was significantly increasing in the order elapsed time after making the model. The degree of luciferase expression was higher in cases of application of in vivo electroporation than in that of non-application and was highest in $100{\sim}150\;V/cm$. In conclusion, in vivo electroporation is effective in transfer and expression of plasmid DNA in normal and ischemic tibialis anterior of rat.

• Journal of Plant Biotechnology
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• 제47권1호
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• pp.66-72
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• 2020

Alstroemeria plants were transformed by using an improved particle-gun-mediated transformation system. Friable embryogenic callus (FEC) induced from the leaves with axil tissues of Alstroemeria plant was used as the target tissue. Also, FEC was transformed with the bar gene was used as a selectable marker. In the case of plasmid pAHC25, 7.5% of the twice-bombarded FEC clumps showed blue foci, whereas the clumps with single bombardment showed only 2.3%. Additionally, a 90° rotation with double bombardment led to a higher frequency (6 times) of luciferase gene expression in PBL9780 than the control treatment. After 8 weeks of bombardment, more than 60 independent transgenic lines were obtained for pAHC25 and nearly 150 independent transgenic lines were obtained for PBL9780, all of which were resistant to PPT and demonstrated either GUS or luciferase activity. Regarding effect of osmotic treatment (0.2 M mannitol) with 7 different periods, the highest transient gene expression was obtained in 8 h before and 16 h after transformation in both pAHC25 and PBL9780. Compared with the control, at least three times more GUS foci and photons were observed in this treatment. With respect to different combinations of mannitol and sorbitol with 8 h before and 16 h after transformation, high numbers of transient and stable transgene expressions were observed in both 0.2 M mannitol and 0.2 M sorbitol used in the osmotic pre-culture. This combination showed the highest transformation efficiency in both pAHC25 (8.5%) and PBL9780 (14.5%). In the control treatment, only 10% of the FEC clumps produced somatic embryos. However, by using 0.2 M mannitol and 0.2 M sorbitol, the frequency of somatic embryos increased to 36.5% (pAHC25) and 22.9% (PBL9780). Of the somatic embryos produced, at least 60% germinated. Approximately 100 somatic embryos from these 210 independent transgenic lines from 2 plasmids developed into shoots, which were then transferred to the greenhouse. PCR analysis confirmed the presence of the bar gene. This is the report on the production of transgenic Alstroemeria plants by using particle bombardment with a high efficiency, thereby providing a new alternative for the transferring of gene of interests in Alstroemeria in the breeding program in the future.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.331-337
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• 2009

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a well-known antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

• Toxicological Research
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• 제20권3호
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• pp.205-211
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• 2004

Concern that some chemicals in our environment may affect human health by disrupt-ing normal endocrine function has prompted a research on interactions of environmental contaminants with steroid hormone receptor. Toxaphene and chlordane are among the 12 persistent organic pollutants identified by the United Nations Environment Programme as requiring urgent attention. We compared the estrogenic activity of two organochlorine pesticides, toxaphene and chlordane, at estrogen receptor a (ER$\alpha$) and estrogen receptor $\beta$ (ER$\beta$). Human hepatoma cells (HepG2) were transiently transfected with rat ER$\alpha$ or ER$\beta$ plus an estrogen-responsive complement C3-luciferase (C3-Luc) reporter gene. After transfection, cells were treated with various concentrations of toxaphene and chlordane to investigate agonism or antagonism of these chemicals. Both toxaphene and chlordane were potent agonists in HepG2 cells for ER$\alpha$. In contrast, these chemicals had a minimal agonist activity with ER$\beta$ and almost abolished 17$\beta$-estradiol-induced ER$\beta$-mediated activity. Therefore, toxaphene and chlordane behaved as an ER$\alpha$ agonist and an ER$\beta$ antagonist with estrogen-responsive reporter plasmid C3-Luc, and exposure to these organochlorine pesticides could have a crictical effect on normal endocrine function.

• 한국융합학회논문지
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• 제13권1호
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• pp.161-166
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• 2022

HIF-2α는 저산소 조건에서 활성화되는 전사인자로 암, 대사증후군, 관절염, 간염 등의 발병 기전에서 핵심 역할을 한다고 보고된 바 있다. 이에 HIF-2α 저해제를 도출하고자 기존 약리활성 구조를 도입한 N'-arylisonicotinolyhydrazide를 골격으로 설정하고 화합물 라이브러리로부터 해당 화합물들을 선택하여 HIF-2α 저해활성을 측정하였다. 이를 위해 HRE-luciferase를 HTB-94세포에 transfection하고 아데노바이러스를 이용하여 HIF-2α를 세포 내로 도입하여 luciferase reporter gene assay를 수행하였다. 2-hydroxy-1-naphthyl 기를 포함한 화합물에서 저해활성이 발견됨에 따라 이 구조를 포함하는 골격을 다시 설정하고 해당 화합물들을 선정하여 활성을 측정하였다. 그 결과 HIF-2α 저해활성과 위양성 시험을 통하여 2 종의 HIF-2α 저해제를 도출하였다. 본 연구는 생물학과 화학의 융합연구로 수행되었으며 도출된 저해제는 후속 저해제 탐색 연구와 HIF-2α의 기능 연구에 활용될 수 있고 관련 질환의 치료제 개발에도 기여 할 것으로 사료된다.

• The Korean Journal of Physiology and Pharmacology
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• 제21권4호
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• pp.449-456
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• 2017

Beauvericin (BEA), a cyclic hexadepsipeptide produced by the fungus Beauveria bassiana, is known to have anti-cancer, anti-inflammatory, and anti-microbial actions. However, how BEA suppresses macrophage-induced inflammatory responses has not been fully elucidated. In this study, we explored the anti-inflammatory properties of BEA and the underlying molecular mechanisms using lipopolysaccharide (LPS)-treated macrophage-like RAW264.7 cells. Levels of nitric oxide (NO), mRNA levels of transcription factors and the inflammatory genes inducible NO synthase (iNOS) and interleukin (IL)-1, and protein levels of activated intracellular signaling molecules were determined by Griess assay, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), luciferase reporter gene assay, and immunoblotting analysis. BEA dose-dependently blocked the production of NO in LPS-treated RAW264.7 cells without inducing cell cytotoxicity. BEA also prevented LPS-triggered morphological changes. This compound significantly inhibited nuclear translocation of the $NF-{\kappa}B$ subunits p65 and p50. Luciferase reporter gene assays demonstrated that BEA suppresses MyD88-dependent NF-${\kappa}B$ activation. By analyzing upstream signaling events for $NF-{\kappa}B$ activation and overexpressing Src and Syk, these two enzymes were revealed to be targets of BEA. Together, these results suggest that BEA suppresses $NF-{\kappa}B$-dependent inflammatory responses by suppressing both Src and Syk.

• 한국축산식품학회지
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• 제38권4호
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• pp.816-822
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• 2018

Bifidobacterium is recognized as one of the most beneficial microorganisms in our gut. Many researches on bifidobacteria have been done to understand their roles in the gut. The objective of the present study was to develop a bioluminescent labelling plasmid vector for bifidobacteria to facilitate their visualization in vitro, in situ, and in vivo. A plasmid replicon (2.0 kb) of plasmid pFI2576 previously identified from B. longum FI10564 was amplified by PCR and cloned into pUC19 plasmid vector (2.68 kb). The cloned replicon was subcloned into pTG262 ($luc^+$) recombinant plasmid vector (7.4 kb) where a luciferase gene ($luc^+$) from pLuc2 (8.5 kb), an Escherichia coli and lactobacilli shuttle vector, was inserted into pTG262 plasmid vector. The final recombinant DNA, pTG262::pFI2576 rep ($luc^+$), was transferred into a B. catenulatum strain. This recombinant strain showed 3,024 relative luminescence units at $OD_{600}$ value of 0.352. Thus, this recombinant plasmid construct can be broadly used for labelling bifidobacteria.

• 대한의생명과학회지
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• 제15권3호
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• pp.179-185
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• 2009

In our previous report, we showed that $PPAR{\gamma}$ does not influence adipogenesis in females with functioning ovaries, indicating that $PPAR{\gamma}$ activity on adipogenesis is associated with sex-related factors. Among the sex-related factors, estrogen has been recognized as a major factor in inhibiting adiposgenesis in females. Thus, we hypothensized that $17{\beta}$-estradiol (E) inhibits 3T3-L1 cell adipogenesis by preventing $PPAR{\gamma}$ activity. E decreased triglyceirde accumulation in differentiated 3T3-L1 cells compared with control group. E also decreased the expression of $PPAR{\gamma}$ mRNA as well as $PPAR{\gamma}$ dependent adipocyte-specific genes, such as adipocyte fatty acid binding protein and tumor necrosis factor $\alpha$. In addition, E not only decreased luciferase reporter activity by $PPAR{\gamma}$, but also transfection of estrogen receptor $\alpha$ ($ER{\alpha}$) or $ER{\beta}$ led to decreases in $PPAR{\gamma}$ reporter gene activation. Moreover, E-activated ERs significantly decreased the luciferase reporter gene activation induced by $PPAR{\gamma}$ transfection, suggesting that estrogen-activated ERs inhibit $PPAR{\gamma}$-dependent transactivation. Accordingly, our results demonstrate that E inhibits the action of $PPAR{\gamma}$ on adipogenesis through E activated ER, providing evidence that lack of estrogen may potentiate $PPAR{\gamma}$ action on adipogenesis.