its activity [10], although at present it remains unknown whether this activity is shared or not with the other members of the Sertad family. A common feature of a number of mouse models deficient for proteins in the Cdk4/cyclinD/E2f pathway is a defective glucose homeostasis due to an impaired maintenance of pancreatic b-cells [5–8]. The expression levels of Sei1, but not of Sertad2, are maximal in pancreatic islets compared to any other tissue examined (Supp. Fig. S1); and, moreover, Sertad2 and Sertad3 mRNA levels are not altered in Sei1-null pancreatic islets. All these observations suggest that pancreatic islets could be more particularly sensitive to the absence of Sei1. Interestingly, Sei1- mice displayed abnormal insulin secretion and blood glucose clearance, as well as a reduction in pancreatic b-cell surface. In addition, pancreatic islets of Sei1-null mice showed increased nuclear staining of the cell cycle inhibitors p27Kip1 and p21Cip1. These two cell cycle inhibitors have been implicated in the negative control of b-cell mass [8]. In particular, overexpression of p27Kip1 in islets results in decreased islet mass and diabetes [27]. Similarly, mice deficient in Skp2, which is responsible for the normal degradation of p27Kip1, present ubiquitous higher levels of p27Kip1, decreased b-cell mass and glucose intolerance, and all these phenotypes are rescued in Skp2/p27Kip1-doubly deficient mice [28]. At present, we do not know why p27Kip1 and p21Cip1 accumulate in the nuclei of Sei1-null b-cells. However, it is tempting to speculate that the absence of Sei1 may increase the availability of Cdk4/D complexes that can interact with p27Kip1 or p21Cip1, thus increasing the amount of Cdk4/D/(p27Kip1-p21Cip1) complexes, which normally reside in the nucleus [12,13]. Although still under debate [29], recent evidence indicates that Cdk4/D/ (p27Kip1-p21Cip1) complexes are inactive [14] and this would be consistent with the decreased islet size of Sei1-null mice. We conclude that Sei1 ablation in mice does not affect tumor growth, but the germline-based strategy followed in this work does not preclude possible effects of acute Sei1 ablation in cancer maintenance. As for the glucose homeostasis, our results suggest that Sei1 may play an important role in diabetes or metabolic disorders. Materials and Methods Ethics Statement Mice were treated in accordance with the Spanish Laws and the Guidelines for Humane Endpoints for Animals Used in Biomed- ical Research, The Spanish National Cancer Research Center (CNIO) is part of the ‘‘Carlos III’’ Health Institute (ISCIII) and all protocols were previously subjected and approved by the Ethical Committee of the ISCIII. Generation of Sei1-Deficient Mice and Animal Care Mice genetically deficient for Sei1 were produced using standard procedures. The construct that eliminates exon 2 of Sei1 (Fig. 1A) was linearized and electroporated into R1 embryonic stem (ES) cells (129Sv). One ES cell clone was used to produce chimaeric male mice, which were crossed with Balb/c females, and the progeny from this cross was genotyped by Southern blotting. All the assays reported in Figs. 1–6 were performed with mice of mixed genetic background Balb/c;129Sv (50:50). All the assays reported in Figs. 7 and 8 were performed in mice that had been backcrossed 9 times with C57BL/6 and therefore had a .99.9% C57BL/6 genetic background. Animals were housed at the CNIO, inside their corresponding pathogen- free barrier areas. All mice were monitored twice per week, and moribund mice were killed humanely. Tumors from non-autolysed Sei1 in Cancer and Diabetes tissues were recovered from moribund or recently deceased mice. Tissue samples were fixed in 10% buffered formalin, embedded in paraffin wax, sectioned at 4 mm and stained with haematoxylin and eosin (H&E) for a detailed histopathological analysis. Antibodies The antibodies used for immunoblotting and immunoprecipi- tation were: Cdk4 (C-22, sc-260), Cdk2 (M-2, sc-163), cyclin D1 (C-20, sc-717), cyclin D2 (M-20, sc-593), p21Cip1 (C-19-G, sc-397- G), and p19Arf (5-C3-1, sc-32748), all from Santa Cruz; and p53 (CM-5), from Novocastra. The antibodies used for immunohisto- chemistry were: p21Cip1 (C-19-G, sc-397-G), from Santa Cruz; p27Kip1 (DCS-72.F6), from Thermo Scientific; insulin (#A564), from Dako; and glucagon (K79bB10), from Sigma Aldrich. Cell Culture MEFs were isolated from embryos at 13.5 days post-coitum from crosses between Sei1(+/2) mice of mixed genetic back- ground Balb/c;129Sv (50:50), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% Fetal Bovine Serum (FBS, Hyclone) and antibiotic/ antimycotic (GIBCO). To obtain splenocytes and thymocytes, spleens were disaggre- gated using a cell strainer (Nunc), and resuspended in complete medium, comprising RPMI supplemented with 100 mM Hepes buffer pH 7.5 (BioWhitaker), 2 mM L-Glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), a commercial mix of non- essential amino acids (BioWhitaker), 0.1% b-mercaptoethanol (Sigma) and antibiotic/antimycotic (GIBCO). Red blood cells were lysed by incubating the preparation for 10 minutes at 4uC in 10 mM KHCO3, 150 mM NH4Cl, 0.1 mM EDTA pH 8, all taken to pH 7.3. Thymocytes were frozen or processed for FACS analysis at this point, whereas splenocytes were plated in complete medium at a density of 26106 cells/ml, activated with 20 mg/ml LPS and 5 mg/ml concanavalin A (Sigma), and counted at different time points. For cell cycle analysis, 26106 were plated in 10 cm diameter dishes and left to reach confluency for 3 days; then, cells were washed 3 times with PBS and cultured in medium without serum for another 3 days. After this time medium with 10% FBS was added, and cells were processed for FACS analysis or protein extraction at different times. For luciferase assays, cells were transfected with pGL3-Basic (Promega) without promoter (basal transcription control) or with the human E2f1 promoter cloned upstream the luciferase reporter. pBabe-E1Fa-Renilla was used as an internal transfection control. Two days later cells were processed using the Dual Luciferase Assay System (Promega) following the manufacturer’s instructions, and firefly and Renilla luciferase activities were measured in a luminometer Glomax (Promega). Firefly luciferase values were normalized to the Renilla control values. For growth curves, 1.56105 cells were plated in 35 mm diameter multiwell plates, cultured in media with different serum concentrations and counted 4 hours later (time point 0), or 2, 4 or 7 days after plating. Serial 3T3 cultivation was done as described [30]. Briefly, 106 cells were plated on 10 cm diameter dishes, 3 days later the total number of cells in the dish were counted, and 106 cells were replated again. This procedure was repeated for 40 passages. The increase in population doubling level (DPDL), was calculated according to the formula DPDL = log(nf/n0)/log2, where n0 is the initial number of cells and nf is the final number of cells. Colony and foci formation assays were performed as described elsewhere [31]. For colonies formation, 66103 cells were PLoS ONE | www.plosone.org 9 January 2010 | Volume 5 | Issue 1 | e8744