open access

https://doi.org/10.11501/3168597

https://hdl.handle.net/2115/51639

分泌型生物発光レポーターを用いた成長ホルモン遺伝子転写活性の連続測定法の開発

日本語

Genetic engineering

2000-03-24

Hokkaido University

24p.

The genetic reporter system has been introduced as a tool for sensitive and convenient monitoring of gene expression. In the present study, the promoter activity of growth hormone (GH) coding gene was sequentially monitored in GH3 cells (rat pituitary adenoma cell-line) by means of bioluminescence which was evoked by a secreted reporter, Vargula hilgendonyfii (VH) luciferase. Three kinds of expression plasmid were constructed, which consists of a rat GH promoter fragment and the VH luciferase cDNA, and transfected to GH3 cells. The sensitivity of reporter was revealed to be extremely high from the dose-response curve of VH luciferase. Using the stable transformant, the reporter activity as well as GH were sequentially measured in plate culture. The intracellular dynamics of reporter signals was analyzed through the response to inhibitors of transcription, protein syntesis and vesicle transport, suggesting that the overall time from transcription to secretion was essentially identical for the VH luciferase and GH although the both proteins took different time course at different intracellular stages. When the GH promoter was activated by triiodothyronine (T3), the reporter activity was increased, and well correlated with the amount of GH mRNA, which indicates a reliability of the reporter system. Furthermore, using the present reporter system, a synergistic effect of T3 and dexamethasone was suggested only on the GH transcription but also on the stability of mRNA. It is concluded that the bioluminescence reporter system using the VH luciferase was an excellent tool for sequential monitoring of the GH transcriptional actlvlty.

2000-03-24

甲第5032号

博士(医学)

博士論文

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