Fbxl21 in the Circadian System Figure 5. In-situ hybridisation for Fbxl3 and Fbxl21 within the SCN of the sheep and mouse. A. Representative images for expression of Fbxl21 (top), Fbxl3 and the neuropeptides, Avp and Vip (bottom) in the hypothalamus of sheep sampled at ZT4-6. Sense controls for Fbxl riboprobes are also shown (top and middle right panels). B. Fbxl21 undergoes circadian expression in the SCN of the mouse. Top: mRNA profiles in the SCN of mice sampled at 6 different time-points (n = 5 per time-point) under either a 12/12 light-dark cycle (LD) or under constant darkness (DD). Data for ZT/ CT0 are double-plotted. Bottom: representative in-situ autoradiograms. Note the SCN-restricted pattern of expression. Two-way ANOVA: time-effect, P,0.001, light condition6time interaction, p = 0.39. C. Fbxl3 does not show circadian expression in the SCN of the mouse. Representative images for expression of Fbxl3 in the brain of mouse sampled at ZT4 and ZT16. Note the ubiquitous pattern of labelling including diffuse expression in the SCN. doi:10.1371/journal.pone.0003530.g005 slaughterhouse (McIntosh Donald, Portlethen, UK). Sheep were killed by the pneumatic captive bolt gun method carried out by a licensed person. Peripheral tissues were sampled from Soay rams killed by an overdose of barbiturate (Euthatal; Rhone Merieux, Essex, UK) administered intravenously. All experiments were performed in accordance with Home Office (UK) legislation and approved by University of Edinburgh Local Ethics Committee. Tissues were rapidly dissected out, frozen onto dry ice and kept at 280uC until sectioning or RNA extraction. Mouse tissue collection Adult male Swiss mice (Charles River, Lyon, France) were kept in a 12-h light/dark cycle (LD) or transferred to constant darkness (DD) for 2 days. After mice were deeply anesthetized with isoflurane and decapitated, brains were removed, frozen and cut in 20-mm coronal sections at the level of the suprachiasmatic region. Five mice were killed every 4 h throughout the LD cycle (expressed in zeitgeber time, ZT0 being the time of lights on) or throughout the DD cycle (expressed in circadian time, CT0 being defined as the projected time of lights on). Experiments were performed in accordance with the rules of the European Committee Council directive of November 24, 1986 (86/609/EEC) and the French Department of Agriculture (license nu67–88 to E.C.). Cloning DNA extraction was done with a QIAamp DNA mini kit (Qiagen). RNA extraction was performed using Tri-reagent (Sigma) according to the manufacturer’s protocol, and cDNA synthesis was carried out using a reverse transcription kit (Qiagen). When sheep EST were not available, ClustalW alignments of mammalian sequences were performed in order to identify conserved regions suitable for primer design (primers from MWG Biotech, Germany). PCR was done with Platinum Taq Hifi (Invitrogen) according to the manufacturer’s protocol. Following agarose gel electrophoresis, PCR fragments of the expected sizes were extracted using a gel extraction kit (Qiagen) and cloned in pGEM-T easy vector (Promega). Four to six positive clones were sequenced (MWG, United Kingdom), and the sequences deposited in GenBank. To generate expression constructs, a second round of PCR was performed using primers flanked by adequate restriction sites and the pGEM-T clone as template. PCR fragments were extracted as described above, digested by the adequate restriction enzymes, purified with a PCR purification kit (Qiagen) and cloned in the expression vector backbone. Two different expression vectors with either a Flag or a 5X-Myc tag were used. To generate promoter reporter constructs, a strategy identical to that described above was applied. All fragments were cloned into the pGL3 basic backbone (Promega) digested with the appropriate restriction enzymes. Sequencing was performed to check accuracy of the re-amplified cloned fragments. All primer sequences and constructs are available upon request. Cell culture, transfection and luciferase assay COS-7 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% Pen/ Strep mix and sodium pyruvate in a humidified atmosphere with 5% CO2 at 37uC. Cells were plated in either 6-well or 24-well plates (for WB or luciferase assay, respectively), and transfected with GeneJuice (Novagen). For luciferase assays, expression vectors were used at different doses depending on the experiment. PLoS ONE | www.plosone.org 6 October 2008 | Volume 3 | Issue 10 | e3530