We can also apply this following system to other research fields. Further modification and development of this system will be needed in order to be applied for more systematic and high throughput screenings. Conclusion Here we present the in vitro real time oscillation monitor ing system. Indeed, we newly found eight can didates out of 299 compounds as circadian entrainment factors. We further con firmed that one of the candidates, 15d PGJ2, triggers the rhythmic expression of endogenous circadian clocks by inducing Crys and Ror, but not Pers, in NIH3T3 cells, indicating that this assay system is a powerful and use ful tool for the initial screening procedure. This system can also be applied not only to find new intracellular mol ecules involved in circadian clocks.
new transcription fac tors, new signaling and degradation pathways, but also to investigate other cellular mechanisms like cell cycle or oncogenesis. Methods Cell culture Rat1 and NIH3T3 fibroblast cells were grown at 37 C and 5% CO2. Rat1 and NIH3T3 cells were grown in Dulbeccos Modified Eagle Medium with L Gln and sodium pyruvate supplemented with 5 and 10% fetal bovine serum, respectively, and antibiotics. Establishment of mPer2 luciferase stably expressing Rat1 cell line A bacterial artificial chromosome clone containing the complete genomic sequence of the mouse Per2 gene was purchased from BACPAC Resource Center at Childrens Hospital Oakland Research Institute. The mPer2 promoter region was isolated and cloned in the pGL3 Basic vector. The mPer2 region spans from 2811 to 110.
Rat1 cells were cotransfected with linearized mPer2 promoter pGL3 and pcDNA3, which contains neomycin resistant gene. Transfection was carried out by using Polyfect Trans fection Reagent according GSK-3 to the manufac tures instructions. The cells were cultured in 10% FBS DMEM containing 500 g ml geneticin for 1 to 2 weeks. Cells were then individually isolated, and 24 clones were established as mPer2 luc Rat1 cells. After screening for the luciferase activity by using IV ROMS, we established two independent clones with clear rhythmic activity. Real time luciferase activity monitoring in living cells mPer2 luc Rat1 cells were seeded in a 35 mm dish at den sity of 2 105 cells and incubated for 2 days. The medium was then exchanged for serum free medium supple mented with a compound to be screened. Compound was diluted to a final concentration of 1 M for peptide and 1 or 10 M for bioactive lipid, respectively. One hour later the medium was replaced with 1% FBS DMEM supple mented with 0. 1 mM luciferin 10 mM HEPES. Light emission was measured and integrated for 1 min at intervals of 15 min with a photomultiplier tube. Data were ana lyzed by LM2400 software.