[Development of a new method for sequential monitoring of transcriptional activities of growth hormone genes by a secreted bioluminescence reporter].
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 hilgendorfii (VH) luciferase. Three kinds of expression plasmids were constructed, which consisted of a rat GH promoter fragment and VH luciferase cDNA, and transfected into GH3 cells. The sensitivity of the reporter was revealed to be extremely high from the dose-response curve of VH luciferase. With the use of the stable transformant, the reporter activity as well as that of GH was sequentially measured in plate culture. The intracellular dynamics of reporter signals was analyzed through the response to inhibitors of transcription, protein synthesis and vesicle transport, which suggested that the overall time from transcription to secretion was essentially identical to those of 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 enhanced, and well correlating with the amount of GH mRNA, which indicated a reliability of the reporter system. Furthermore, in the present reporter system, a synergistic effect of T3 and dexamethasone was observed not only on the GH transcription but also on the stability of mRNA. It is concluded that the bioluminescence reporter system using the VH luciferase is an excellent tool for sequential monitoring of the GH transcriptional activity.
Tanahashi
Y
Y
Department of Physiology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan.
D000818:Animals; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter; D013006:Growth Hormone / Q000235:genetics*; D006801:Humans; D008156:Luciferases; D008163:Luminescent Measurements; D011401:Promoter Regions, Genetic; D051381:Rats; D015533:Transcriptional Activation
D013006:Growth Hormone; D008156:Luciferases