Based on the positive role of Sei1 on Cdk4/D and E2f1 activities, it is conceivable that Sei1 could have a role in cancer and/or pancreatic islet biology. With regard to cancer, amplifi- cation of the genomic locus of Sei1 has been found in human head and neck squamous cell carcinomas, ovarian carcinomas and gastric carcinoma [16–18]. Here we have tested the current models on Sei1 function by generating and analyzing Sei1- deficient cells and mice. Results Sei1-Null Mice Are Viable and Have a Normal Longevity The Sei1 gene is composed by two exons, the first one is non- coding and the second one contains the complete open reading frame. We engineered a gene-targeting vector that replaced exon 2 of Sei1 with a neomycin resistance cassette (Fig. 1A). Targeted ES clones were confirmed by Southern blot analysis (Fig. 1B) and were used to obtain Sei1-deficient mice (see below). In addition, we generated Sei1-null ES cells by culturing Sei1-heterozygous ES cells in medium with high neomycin concentrations (see Materials and Methods; Fig. 1B). Complete Sei1 deficiency was corroborat- ed by Northern blot analysis of Sei1-null ES cells (Fig. 1C) and qRT-PCR from Sei1-deficient MEFs (Fig. 1D). Sertad2 is the closest homolog of Sei1, and we wondered whether inactivation of Sei1 had an impact on the expression of Sertad2 in MEFs. Sei1 in Cancer and Diabetes Analysis of Sertad2 expression indicated that its levels remained unchanged in Sei1-het and Sei1-null MEFs (Fig. 1D). Homozygous Sei1-null mice were viable, fertile and showed no overt pathologies. We checked the expression of Sei1 and Sertad2 in a variety of organs by qRT-PCR. Both Sei1 and Sertad2 were expressed in most organs examined (Supp. Fig. S1). Detailed histopathological analyses did not reveal abnormalities in Sei1-null tissues (data not shown). When wt, Sei1-het and Sei1-null cohorts of mice were aged, their survival curves were indistinguishable (Supp. Fig. S2), as well as the pathologies observed in moribund old mice (see below the incidence of spontaneous cancer). Sei1-Null Cells Proliferate Normally Mouse embryo fibroblasts (MEFs) were obtained from wild type, Sei1-het and Sei1-null mice. First, we studied Cdk4 complexes in asynchronously growing cells, but we did not observe differences in the abundance of Cdk4/D1 or Cdk4/D2 complexes (Fig. 2A and Supp. Fig. S3A). To check whether the absence of Sei1 could affect the cell cycle, we rendered wt and Sei1-null MEFs quiescent by serum withdrawal, and triggered cell cycle re-entry by serum addition. As shown in Fig. 2B, Sei1-null MEFs entered into S phase with normal efficiency and kinetics. As a control, Cdk4-null MEFs presented a significant reduction in the percentage of cells entering S phase, as previously reported [19]. To examine in further detail the cell cycle, we checked the Figure 1. Generation of Sei1-deficient mice. (A) Strategy used to eliminate the second exon of Sei1, which contains the entire open reading frame for Sei1. (B) Southern blot confirming the ablation of Sei1 alleles in ES cells. (C) Northern blot confirming the loss of Sei1 transcription in Sei1- ES. (D) Levels of Sei1 and Sertad2 transcripts in primary MEFs (n = 3, per genotype). Values correspond to average and s.d. of the levels relative to actin. doi:10.1371/journal.pone.0008744.g001 PLoS ONE | www.plosone.org 2 January 2010 | Volume 5 | Issue 1 | e8744