Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase.
The promoter activity of growth hormone (GH) was continuously monitored in rat pituitary adenoma cells (GH3) by a secreted bioluminescence reporter, Vargula hilgendorfii (Vh) luciferase. The sensitivity of the reporter was approximately 60-times higher than that of the firefly luciferase. GH3 cells were transfected with a plasmid containing a DNA sequence of the GH promoter (1.8 kb) and a full length of the Vh luciferase cDNA. Using the stable transformants, the Vh luciferase activity was monitored in the plate culture through the bioluminescence of Vh luciferase secreted into the culture medium. The reporter activity was well correlated with GH mRNA as well as GH when the GH promoter activity was activated by thyroid hormone. To develop a continuous monitoring system of the promoter, the reporter activity was sequentially measured in the perfusion system. When the promoter activity of the stable transformants was suppressed by a transcription inhibitor, the reporter activity and GH in the perfusate were simultaneously decreased. The Vh luciferase reporter is a sensitive and convenient tool for continuous and prolonged measurement of promoter activity in living cell culture systems.
Tanahashi
Y
Y
Department of Physiology, Hokkaido University Graduate School of Medicine, N15 W7 Kita-ku, Sapporo 060-8638, Japan.
Ohmiya
Y
Y
Honma
S
S
Katsuno
Y
Y
Ohta
H
H
Nakamura
H
H
Honma
K I
KI
D000818:Animals; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D015152:Blotting, Northern; D002166:Camptothecin / Q000494:pharmacology; D002478:Cells, Cultured; D003530:Cyprinidae / Q000235:genetics / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D007124:Immunoenzyme Techniques; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D008957:Models, Genetic; D010477:Perfusion; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D013963:Thyroid Hormones / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D018931:Antineoplastic Agents, Hormonal; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D013963:Thyroid Hormones; D012313:RNA; D003907:Dexamethasone; D008156:Luciferases; D002166:Camptothecin