Real-time monitoring of circadian clock oscillations in primary cultures of mammalian cells using Tol2 transposon-mediated gene transfer strategy.
BACKGROUND
The circadian rhythm in mammals is orchestrated by a central pacemaker in the brain, but most peripheral tissues contain their own intrinsic circadian oscillators. The circadian rhythm is a fundamental biological system in mammals involved in the regulation of various physiological functions such as behavior, cardiovascular functions and energy metabolism. Thus, it is important to understand the correlation between circadian oscillator and physiological functions in peripheral tissues. However, it is still difficult to investigate the molecular oscillator in primary culture cells.


RESULTS
In this study, we used a novel Tol2 transposon based Dbp promoter or Bmal1 promoter driven luciferase reporter vector system to detect and analyze the intrinsic molecular oscillator in primary culture cells (mouse embryonic fibroblasts, fetal bovine heart endothelial cells and rat astrocytes). The results showed circadian molecular oscillations in all examined primary culture cells. Moreover, the phase relationship between Dbp promoter driven and Bmal1 promoter driven molecular rhythms were almost anti-phase, which suggested that these reporters appropriately read-out the intrinsic cellular circadian clock.


CONCLUSIONS
Our results indicate that gene transfer strategy using the Tol2 transposon system of a useful and safe non-viral vector is a powerful tool for investigating circadian rhythms in peripheral tissues.
Yagita
Kazuhiro
K
Department of Neuroscience and Cell Biology, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita, Osaka, 565-0871 Japan. kyagita@anat1.med.osaka-u.ac.jp
Yamanaka
Iori
I
Emoto
Noriaki
N
Kawakami
Koichi
K
Shimada
Shoichi
S
D000818:Animals; D001683:Biological Clocks / Q000235:genetics*; D002417:Cattle; D002478:Cells, Cultured; D002940:Circadian Rhythm / Q000235:genetics*; D005822:Genetic Vectors; D051379:Mice; D011401:Promoter Regions, Genetic; D051381:Rats; D018626:Retroelements; D014162:Transfection
D018626:Retroelements