post-hoc Tukey test. Differences were considered to be statistically significant when P,0.05. Results PDL Induces Survivin Expression and Promotes Beta-cell Mass Expansion In C57BL/6 mice, the duct leading to the pancreatic tail was closed while the organ’s head located adjacent to the stomach and duodenum remained unaffected. Within two weeks, the beta-cell mass in the ligated part of pancreas increased more than 2-fold following surgery, and beta-cell proliferation increased 9-fold determined by ki67 staining (Fig. 1a). No difference in beta-cell mass and proliferation was found in the non-ligated portion (head) of the pancreas (Fig. 1a). Previous reports showed survivin to be present in mouse pancreatic islets transiently during embryonic development and neonatal period, and become undetectable by 2-month of age [20,21]. We found that survivin was induced in the islets of the ligated tail of pancreas within 2 weeks after PDL,reaching its peak at PDL day 7 (Fig. 1b). Immunofluorescent staining showed that Survivin Regulation of Beta-Cell Mass Expansion survivin was not detectable in the whole pancreas in the tail of day 7 sham-operated pancreas, but regained its expression throughout the pancreatic epithelium including islet beta cells in the tail of pancreas7 days after PDL (Fig. 1c). Generation of Beta-cell-specific Survivin-deficient Mice To investigate the essential role of survivin induction in the expansion of pancreatic beta-cell mass, we generated a rat insulin promoter (RIP)-driven survivin knockout mouse by Cre-loxP recombination system. The resultant RIPcre+survivinfl/fl mice had deletion of survivin specifically in pancreatic beta cells and efficient deletion in the islets in both sexes (Fig. 2a–d). PCR analyses confirmed the presence of the deleted allele specifically in islets of RIPcre+survivinfl/flmice (Fig. 2c). The expression of survivin in other tissues, including the liver and kidney, was unaffected (Fig. 2d). RIPcre+survivinfl/fl mice were born at expected Mendelian frequencies and survived past weaning. Because of the lack of survivin expression during embryonic development and neonatal period, the mutant mice exhibited a decreased beta-cell mass at 2- month of age as compared to controls. The male mutant mice had Figure 1. PDL activates survivin expression and promotes beta-cell mass expansion. (a) PDL markedly increased beta-cell mass (left panel) and beta-cell proliferation (right panel) in the tail part of pancreas (black bars) at day 3, 7 and 14 as compared to the unligated head of the same pancreas (gray bars) and the tail of a sham-operated pancreas (white bars) (n = 5 at each time point). **P,0.01. (b) Western blots (left panel) and quantification (right panel) show that survivin expression is detected in the ligated tail of 8-week-old mouse pancreas from PDL day3 to 14 (n = 5 at each time point). Compared to D3, **p,0.01. (c) Immunofluorescent staining shows that survivin is not expressed in the whole pancreas in the tail of day 7 sham-operated pancreas, but reexpressed throughout the pancreatic epithelium including islet beta cells in the tail of pancreas at PDL day 7 (original magnification, 6200). The data represent as means 6 SE. doi:10.1371/journal.pone.0041976.g001 PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e41976