• Asian-Australasian Journal of Animal Sciences
• /
• 제11권5호
• /
• pp.498-502
• /
• 1998

The present study was designed, fIrst to develop the new methodology to measure the bioluminescence activity easily in live developing bovine embryos by photoncounting, and secondly to compare the expression efficiency of four luciferase reporter genes in bovine embryos at four- to 16-cell stages. In experiment 1, equimolar pSVlacZ and pSVEluc were microinjected into the pronucleus of fertilized bovine oocytes. At 2 days after micro injection, bioluminescence activity of these embryos was measured by photoncounting with a luminometer for 1 min, and lacZ gene expression in the same embryos was assayed by X-gal staining. All the luciferase-positive oocytes showed some bacterial ${\beta}$-galactosidase activity irrespective of the intensity. In experiment 2, four firefly luciferase genes (pTKEluc, pTK6WEluc, pSVEluc and pMiwluc) were introduced by micro injection, and the injected embryos were cultured for the following 2 days. Detection of the luciferase gene expression was done by photoncounting at 5 to 55 min. Over the measurement period, the luciferase activity was almost constant irrespective of the transgenes microinjected. The luciferase activity and expression efficiency at 2 days after microinjection were not significantly affected by the difference in the microinjected transgenes. The present results demonstrated that the bioluminescence activity in live developing bovine embryos could be measured quickly by photoncounting.

• Journal of Microbiology
• /
• 제38권4호
• /
• pp.255-259
• /
• 2000

For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral activities of three known compounds and the result was compared with results from the conventional plaque assay for measuring the production of infectious viruses. When human fibroblast cells were infected with HCMV/pp28-luc, luciferase activity was observed at 2 days after infection and reached maximum at 6 days after infection, whereas the production of infectious virus was maximal at 4 days after infection. The antiviral activities of ganciclovir, acyclovir, and papaverine were measured in HFF cells infected with HCMV/PP28-luc and the luciferase activity was compared with the infectious virus titers. Luciferase activity decreased as the concentration of ganciclovir or papaverine increased, while there was a slight decrease in luciferase activity with acyclovir. The level of the decrease in Luciferase activity was comparable to the level of decrease in the production of infectious virus. Therefore, the antiviral assay using recombinant virus HCMV/pp28-luc resulted in sensitivity similar to the conventional plaque assay with a significant reduction in assay time.

• 미생물학회지
• /
• 제38권4호
• /
• pp.255-255
• /
• 2002

For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral activities of three known compounds and the result was compared with results from the conventional plaque assay for measuring the production of infectious viruses. When human fibroblast cells were infected with HCMV/pp28-luc, luciferase activity was observed at 2 days after infection and reached maximum at 6 days after infection, whereas the production of infectious virus was maximal at 4 days after infection. The antiviral activities of ganciclovir, acyclovir, and papaverine were measured in HFF cells infected with HCMV/PP28-luc and the luciferase activity was compared with the infectious virus titers. Luciferase activity decreased as the concentration of ganciclovir or papaverine increased, while there was a slight decrease in luciferase activity with acyclovir. The level of the decrease in Luciferase activity was comparable to the level of decrease in the production of infectious virus. Therefore, the antiviral assay using recombinant virus HCMV/pp28-luc resulted in sensitivity similar to the conventional plaque assay with a significant reduction in assay time.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제29권2호
• /
• pp.185-192
• /
• 2014

Recombinant proteins can be generated quickly and easily in large amounts and at low-cost in silkworm larvae by using Bombyx mori nuclear polyhedrosis virus (BmNPV). We searched for high-permissive silkworm strains that have high production levels of heterologous proteins and are thus suitable for use as biofactories. In this study, we performed the analysis using a BmNPV vector expressing luciferase as a marker, and we confirmed protein expression by evaluating luciferase activity, determined by western blotting and luciferase ELISA, and confirmed transcription expression by semi- and quantitative real time PCR. For the selection of host silkworm strains, we first chose 52 domestic BmNPV sensitive strains and then identified 10 high-permissive and 5 low-permissive strains. In addition, to determine which hybrid of the high-permissive strains would show heterosis, nine strains derived through three-way crossing were tested for luciferase activity by western blotting, and luciferase ELISA. We found a correlation between luciferase activity and luciferase protein expression, but not transcription. There was no noticeable difference in protein expression levels between Jam313 as the high-permissive control strain and the three-way hybrid strains; however, the three-way cross strains showed lower luciferase activity compared with Jam313. In this study, luciferase protein production in the larvae of 52 domestic silkworm strains was elucidated using BmNPV.

• Preventive Nutrition and Food Science
• /
• 제8권1호
• /
• pp.61-65
• /
• 2003

This study was investigated to identify the regulatory mechanism of ACC gene expression by hormones and nutrition. The fragment of ACC promoter I (PI) -220 bp region was recombined to pGL3-Basic vector with luciferase as a reporter gene. The primary hepatocyte from the rat was used to investigate the regulation of ACC PI activity. ACC PI (-220 bp)/luciferase chimeric plasmid was transfected into primary rat hepatocyte by using lipofectin. ACC PI activity was shown by measuring luciferase activity. The addition of insulin, dexamethasone, and triiodothyronine to the culture medium increased the activity of ACC PI by 2.5-, 2.3- and 1.8-fold, respectively. In the presence of 1 $\mu$M dexamethasone, the effects of insulin was amplified about 1.2-fold showing the additional effects of dexamethasone. Moreover the activity of luciferase was increased by insulin, dexamethasone, and triiodothyronine treatment approximately 4-fold. These results indicated that insulin, dexamethasone and thyroid hormone coordinately regulate ACC gene expression via regulation of promoter I activity. On the -220 to ＋21 region of ACC PI, the addition of the glucose to the culture medium increased the activity of ACC PI. With 25 mM glucose, luciferase activity increased by 7-fold. On the other hand, on the -220 bp region, ACC PI activity was not changed by polyunsaturated fatty acids. Therefore, it can be postulated that there are response elements for insulin, triiodothyronine, dexamethasone, and glucose, but not PUFAs on the -220 bp region of ACC PI.

• Biomolecules & Therapeutics
• /
• 제7권4호
• /
• pp.315-321
• /
• 1999

Since it has been known that hypoxia increases inducible nitric oxide synthase (iNOS) gene expression through hypoxia responsive element, it was possible to establish the hypothesis that nitric oxide could be a mediator of hypoxia to inhibit Cyplal promoter activity. In order to test this hypothesis, we have undertaken the study to examine the effects of hypoxia and nitric oxide on Cyplal promoter activity in Hepa I cells. Mouse Cyplal 5'flanking DNA, 1.6 Kb was cloned into pGL3 expression vector in order to construct pmCyplal-Luc. Hepa I cells were transfected with pmCyplal-Luc and were treated with $10^{-9}$ M TCDD and nitric oxide producing agents, such as lipopolysaccharide(LPS), sodium nitroprusside (SNP). Luciferase activity of reporter gene was measured from pmCyplal-Luc transfected Hepa I cell lysate which contains 2 g total protein using luciferin as a substrate. Nitric oxide producing agents, such as lipopolysaccharide (LPS), sodium nitroprusside(SNP) showed inhibition of luciferase activity that was induced by $10^{-9}$M TCDD treatment with dose dependent manner. Concomitant treatment of 1mM $N^G$-nitro-ι-arginine with $10^{-6}$~$10^{-4}$M sodium nitro-prusside recovered luciferase activity from the TCDD induced luciferase activity that was inhibited by nitric oxide producing agents. These demonstrated that nitric oxide could be a mediator of inhibitors on dioxin induced Cyplal expression in Hepa I cells.

• Journal of Ginseng Research
• /
• 제21권3호
• /
• pp.141-146
• /
• 1997

The role of cAMP in the differentiation process of F9 cells induced by ginsenosides was examined by performing transient transfixion assay with CRE-luciferase reporter plasmid, GR thansactivation assay with GRE-luciferase activity with or without treatment of CAMP and forskolin, an activator of adenylate cyclase, and protein klnase A assay in the presence of ginsenosides. Ginsenosides had no effect on CRE-transactivation activity, whereas retinoic acid induced the activity. When cAMP or forskolin was treated with ginsenosides, GRE-luciferase activity was further augumented by them. In addition, ginsenosides induced protein kinase A activity in the presence of cAMP. These results suggest that ginsenosides activate cAMP-dependent protein kinase A which, in turn, increase GR activity in F9 cells.

• 자연과학논문집
• /
• 제15권1호
• /
• pp.89-95
• /
• 2005

폐암을 일으키는 것으로 알려진 JSRV는 NIH3T3 세포를 transformation 시키는 성질이 있다는 것으로 알려져 있다. 이 바이러스 중 Envelope 단백질이 NIH3T3를 transformation시키는 것으로 알려져서 이것이 세포내에서 어떤 전사인자를 활성화시키는지를 luciferase 리포터 플라스미드를 이용한 transient transfection 방법으로 조사하였다. 그 결과 Envelope 단백질은 NF-kB와 AP-1의 활성을 높이는 것으로 밝혀졌다.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
• /
• pp.140-140
• /
• 1998

The aim of this study was to find out the effect of hypoxic condition on the regulation of cyplal gene expression. pcyplal-Luc construct was cloned and transfected into Hepa I cells. When Hepa-I cells containing pcyplal-Luc were treated by DFO (desferrioxamine) which is iron-chelating agent, the stimulatory effect of luciferase by TCDD was decreased. This inhibitory effect of desferrioxamine on the luciferase activity was dose dependent and abolished by concomitant treatment with N$\^$G/-nitro-ι-arginine. And when cobalt chloride which is known as a hypoxia inducing chemical was administrated, the stimulatory effect of luciferase by TCDD was also decreased. This inhibitory effect of cobalt chloride on the luciferase activity was dose dependent and abolished by concomitant treatment with N$\^$G/-nitro-ι-arginine. These data showed that hypoxic condition down regulates cyplal gene expression and this might be through nitric oxide action.

• 생명과학회지
• /
• 제23권2호
• /
• pp.167-174
• /
• 2013

Peroxiredoxin 6 (Prx 6)는 티올-특이적 항산화 단백질에 속하는 효소로서 산화적 스트레스로부터 세포를 보호할 뿐만 아니라 과산화물의 환원작용을 촉매한다. 본 연구에서는 인간 Prx 6의 promoter를 이용하여 항산화반응을 유발하는 추출물을 효과적으로 스크리닝하는 새로운 형질전환마우스를 개발하는 최종목적을 달성하기 위한 중간단계로서, hPrx 6/Luc 벡터를 개발하고, 이들 벡터의 안정적 발현과 성공적 반응성을 세포주를 이용하여 확인하고자 하였다. 이를 위해, 인간 Prx 6 promoter를 증폭하여 luciferase cDNA와 결합한 hPrx 6/Luc 벡터를 제조하였으며, 제조된 벡터를 제한효소 절단과 염기서열분석을 통해 확인하였다. hPrx 6/Luc 벡터는 NCI-H460 세포에 transfection한 후 인삼(KWG), 홍삼(KRG), 맥문동(LP), 홍문동(RLP)의 4가지 추출물을 처리하여 luciferase activity를 측정하였다. 그 결과, luciferase activity는 4가지 추출물에 의해 효과적으로 증가하였고, 특히 KRG과 LP를 처리한 그룹이 KWG과 RLP를 처리한 그룹보다 높았다. 또한, luciferase activity는 RLP 농도에 의존적으로 증가하였다. hPrx 6/Luc 벡터와 hPrx 6 mRNA반응의 차이를 비교하기 위해, 4가지 추출물을 처리한 후 hPrx 6 mRNA의 양을 RT-PCR로 분석하였다. 그러나, hPrx 6 mRNA의 양은 비록 고농도의 RLP 추출물에서는 약간의 증가가 관찰되었지만, 대조군에 비하여 4가지 추출물에서 유의적인 차이가 없었다. 한편, 4가지 추출물에 의한 superoxide dismutase (SOD) 활성의 변화는 비록 일부 차이는 있었지만 hPrx 6/Luc 벡터와 유사한 반응을 나타내었다. 따라서, 이러한 결과는 hPrx 6/Luc 벡터는 성공적으로 제조되었고, 세포내에서 안정적으로 발현하면서 항상화물질에 민감하고 정량적으로 반응할 수 있음을 제시하고 있다. 더불어, 이러한 세포주에서 확인결과를 바탕으로 형질전환마우스가 개발된다면, 항산화물질을 정량적으로 스크리닝하는 시스템으로 적용가능성이 매우 높음을 보여주고 있다.