• Molecules and Cells
• /
• 제42권11호
• /
• pp.783-793
• /
• 2019

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of $ATF6{\alpha}$ by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

• 대한본초학회지
• /
• 제28권2호
• /
• pp.39-44
• /
• 2013

Objectives : Gambigyeongsinhwan 16 (GGEx16), gambigyeongsinhwan 18 (GGEx18) and gambitongseong capsule are shown to be involved in the regulation of obesity. Therefore, the aim of this study was to compare the reporter activity of anti-obesity genes such as peroxisome proliferator-activated receptor ${\alpha}$ ($PPAR{\alpha}$) and $PPAR{\delta}$ by GGEx16, GGEx18 and gambitongseong capsule. Methods : After NMu2Li liver cells, C2C12 skeletal muscle cells and 3T3-L1 preadipocytes were treated with GGEx16 (1 ${\mu}g/ml$), GGEx18 (1 ${\mu}g/ml$) and different concentrations of gambitongseong capsule, the transactivation of $PPAR{\alpha}$ and $PPAR{\delta}$ was measured by a luciferase reporter gene assay. Results : $PPAR{\alpha}$ reporter gene activity in NMu2Li liver cells and 3T3-L1 preadipocytes was significantly increased by GGEx16, GGEx18 and gambitongseong capsule compared with control, whereas $PPAR{\alpha}$ reporter gene activity in C2C12 skeletal muscle cells was significantly increased by GGEx18 only compared with control. Similarly, $PPAR{\delta}$ reporter gene activity in 3T3-L1 preadipocytes was also significantly increased by GGEx18 compared with control. $PPAR{\delta}$ reporter gene activity in C2C12 skeletal muscle cells was significantly increased by GGEx16 and GGEx18 compared with control although $PPAR{\delta}$ reporter gene activity in NMu2Li liver cells was not changed by these three formulas. Conclusions : These results suggest that all three formulas have the ability to stimulate $PPAR{\alpha}$ and $PPAR{\delta}$ transactivation in animal cell lines with high metabolic rates. In particular, this effects were most prominent in GGEx18-treated cells. In addition, it is likely that GGEx18 may be used as an effective anti-obesity composition.

• Journal of Microbiology and Biotechnology
• /
• 제17권10호
• /
• pp.1727-1732
• /
• 2007

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAA-CoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.

• 한국수산과학회지
• /
• 제46권6호
• /
• pp.901-906
• /
• 2013

We examined the availability of the lacZ gene (${\beta}$-galactosidase gene) as a reporter of foreign gene transfer in the cysts of Artemia franciscana (A. franciscana) to conduct a risk assessment of living genetically modified organisms (LMOs) in the marine ecosystem. The LacZ gene was transferred to decapsulated cysts by particle bombardment, and its insertion and expression were assessed by means of polymerase chain reaction (PCR) and X-gal staining. X-gal staining indicated lacZ expression in all A. franciscana examined (including the control group), which exhibited not only negative but also positive PCR amplification. Endogenous ${\beta}$-galactosidase is highly active in the whole body of A. franciscana during all stages of the life cycle. Thus, the lacZ gene is unsuitable as a reporter for foreign gene transfer in A. franciscana cysts, because it is difficult to discriminate between exogenous and endogenous ${\beta}$-galactosidase activity.

• 대한핵의학회지
• /
• 제38권2호
• /
• pp.175-179
• /
• 2004

Molecular nuclear cardiac imaging has included Tc-99m Annexin imaging to visualize myocardial apoptosis, but is now usually associated with gene therapy and cell-based therapy. Cardiac gene therapy was not successful so far but cardiac reporter gene imaging was made possible using HSV-TK (herpes simplex virus thymidine kinase) and F-18 FHBG (fluoro-hydroxymethylbutyl guanine) or I-124 FIAU (fluoro-deoxyiodo-arabino-furanosyluracil). Gene delivery was performed by needic injection with or without catheter guidance. Tk expression did not last longer than 2 weeks in myocardium. Cell-based therapy of ischemic heart or failing heart looks promising, but biodistribution and differentiation of transplanted cells are not known. Reporter genes can be transfected to the stem/progenitor cells and cells containing these genes can be transplanted to the recipients using catheter-based purging or injection. Repeated imaging should be available and if promoter are varied to let express reporter transgenes, cellular (trans)differentiation can be studied. NIS (sodium iodide symporter) or D2R receptor genes are promising in this aspect.

• BMB Reports
• /
• 제55권4호
• /
• pp.187-191
• /
• 2022

Obesity is caused by an imbalance between energy intake and energy expenditure. Exercise is attracting attention as one of the ways to treat obesity. Exercise induces 'beige adipogenesis' in white adipose tissue, increasing total energy expenditure via energy dissipation in the form of heat. Also, beige adipogenesis can be induced by treatment with a beta-adrenergic receptor agonist. We developed a Cidea-dual reporter mouse (Cidea-P2A-Luc2-T2A-tdTomato, Luciferase/tdTomato) model to trace and measure beige adipogenesis in vivo. As a result, both exercise and injection of beta-adrenergic receptor agonist induced beige adipogenesis and was detected through fluorescence and luminescence. We confirmed that exercise and beta-adrenergic receptor agonist induce beige adipogenesis in Cidea-dual reporter mouse, which will be widely used for detecting beige adipogenesis in vivo.

• Molecules and Cells
• /
• 제26권2호
• /
• pp.131-139
• /
• 2008

Expression of the seven open reading frames (ORFs) of single-stranded DNA Curtoviruses such as Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV) is driven by a bi-directional promoter. To investigate this bidirectional promoter activity with respect to viral late gene expression, transgenic Arabidopsis plants expressing a GUS reporter gene under the control of either the BCTV or BSCTV bi-directional promoter were constructed. Transgenic plants harboring constructs showed higher expression levels when the promoter of the less virulent BCTV was used than when the promoter of the more virulent BSCTV was used. In transgenic seedlings, the reporter gene constructs were expressed primarily in actively dividing tissues such as root tips and apical meristems. As the transgenic plants matured, reporter gene expression diminished but viral infection of mature transgenic plants restored reporter gene expression, particularly in transgenic plants containing BCTV virion-sense gene promoter constructs. A 30 base pair conserved late element (CLE) motif was identified that was present three times in tandem in the BCTV promoter and once in that of BSCTV. Progressive deletion of these repeats from the BCTV promoter resulted in decreased reporter gene expression, but BSCTV promoters in which one or two extra copies of this motif were inserted did not exhibit increased late gene promoter activity. These results demonstrate that Curtovirus late gene expression by virion-sense promoters depends on the developmental stage of the host plant as well as on the number of CLE motifs present in the promoter.

• 한국양식학회지
• /
• 제7권1호
• /
• pp.41-54
• /
• 1994

E. coli의 \beta-galactosidase$ 유전자를 미꾸라지 수정난에 미세현미 주입하고 이를 분석함으로써 미꾸라지에 외래 유전자 이식을 위한 reporter 유전자로서의 유용성을 검토하였다 X-gal 염객분석, 4-methylumbelliferyl-$\beta$-D-galactoside (MUG) 분석을 수행한 결과 유전자 이식 처리군 및 대조군에서 모두 \beta-galactosidase$의 활성이 관찰되었으며 PCR, dot blot 및 southern blot분석결과 역시 유전자 이식 처리군과 대조군에서 모두 유사한 양상을 나타내었다. 처리군 및 대조군의 PCR product의 염기서열은 E. coli의 \beta-galactosidase$ 유전자와 매우 높은 homology를 갖고 있었으며 pH에 따른 X-gal 염색 분석을 수행한 결과 미꾸라지에 관찰되는 본 효소는 pH 4.5에서 가장 높은 활성을 나타내었다. 따라서 앞으로 미꾸라지를 대상으로 한 외래 유전자 이식시 E. coli의 \beta-galactosidase$ 유전자의 reporter 유전자로서의 사용은 신중한 재검토가 이루어져야만 할 것으로 판단된다.

• 미생물학회지
• /
• 제36권1호
• /
• pp.1-7
• /
• 2000

발광 박테리아인 Photobacterium 종들의 lux 오페론 하부 영역에서 riboflavin 생합성에 관여하는 유전자들(ribⅠ,Ⅱ,Ⅲ,Ⅳ)이 발견되었다. Photobacterium phosphoreum의 lux 유전자와 rib 유전자를 포함하는 intergenic 영역의 단일사슬 DNA가 P. phosphoreum의 mRNA에 의하여 S1 nuclease digestion에서 손상받지 않았으며, ribⅠ에 의하여 암호화되는 P. phosphoreum의 riboflavin synthase의 활성도가 lux-specific한 효소들인 luciferase 혹은 fatty acid reductase 활성도와 같이 bioluminescence intensity의 발현과 함께 대수기 말기에서 증가하는 박테리아 발광반응의 특이한 조절 체계인 'autoinduction' 양상을 보였다. 또한 P. leiognathi의 luxB로부터 ribⅡ까지 포함하는 DNA를 강력한 lux 프로모터와 reporter(chloramphenicol acetyl transferase, CAT) 유전자 사이에 삽입하고 접합(conjugation)의 방법으로 P. leiognathi에 유전자 전이(gene transfer)시켜 CAT reporter 유전자의 발현을 P. leiognathi에서 조사한 바, 그 유전자의 발현 정도에 큰 차이가 없었을 뿐만 아니라 이 구조에서 lux 프로모터를 제거하게 되면 CAT reporter 유전자의 발현이 전혀 나타나지 않았다. 이들 실험 결과들은 lux 유전자와 rib 유전자의 intergenic영역에 lux 오페론의 전사 종결 구조(transcriptional terminator)가 존재하지 않으며 ribflavin 생합성 유전자들이 그들 고유의 프로모터에 의하여 전사되는 것이 아니라 lux 오페론의 프로모터에 의하여 발현됨을 나타내는 것으로, 이는 Photobacterium 종들에서 lux 유전자와 rib 유전자들은 공동의 발현 조절 체계를 갖는 것으로 요약된다.

• 한국해양바이오학회지
• /
• 제2권2호
• /
• pp.81-85
• /
• 2007

글루코코르티코이드의 다양한 생리학적 과정은 이 호르몬에 의해 활성화된 수용체가 표적 유전자의 전사를 촉진 혹은 억제시킴으로써 일어나게 된다. 본 논문은 렌티바이러스 리포터 시스템을 이용하여 글루코코르티코이드 호르몬에 의한 GR 활성을 핵내에서 GRE에 의해 유도된 리포터 단백질인 mRFP 또는 루시퍼라아제의 발현을 통해 정성, 정량화 하였다. 그 결과 GR이 endogenous 하게 발현되는 HeLa 세포에서 코티졸을 처리하였을 때 활성화된 GR에 의해 GRE-inducible한 RFP와 루시퍼라아제의 발현이 각각 공초점 형광 현미경과 IVIS-200을 이용하여 형광 또는 BLI을 통해 증가함을 확인하였다. 이러한 결과를 통해 렌티바이러스 리포터 시스템을 이용한 연구는 세포 내에서 뿐 만 아니라 향후 생체내에서의 GR signaling을 모니터링하는데 유용하게 사용되어질 수 있을 것이다.