Role of Wdr13 Gene in Beta Cell Proliferation Figure 1. Generation of Wdr13 knockout mice. A) Exon2 and exon3 (partial) of Wdr13 gene were replaced by neomycin resistance marker gene. B) Southern blot analysis showing 9.3 kb EcoRI fragment from the wild type allele and 3.8 kb fragment from the mutant allele using a 700 bp probe from 59 end of the locus. C) Northern blot analysis by Wdr13 cDNA probe showing the absence of Wdr13 transcript in Wdr132/0 mice (upper panel) and ethidium bromide staining as loading control (lower panel). D) Western blot analysis from testis, pancreas and purified islets using anti-WDR13 rabbit polyclonal antibody (upper panel) showing the absence of WDR13 protein in Wdr132/0 mice, and anti-beta actin as loading control (lower panel). The expression of WDR13 is extremely high in islets in comparison to acinar cells. E) Expression of WDR13 protein in insulin sensitive tissues such as liver, pancreas, muscle and adipose tissues using anti WDR13 antibody (upper panel) and posceau stained blot as loading control (lower panel). (+/0 wild type male; 2/0 knockout male). doi:10.1371/journal.pone.0038685.g001 Overexpression of Wdr13 gene results in cell growth retardation To further understand the role of Wdr13 gene in beta cell proliferation, WDR13 protein was overexpressed in MIN6 cell line using pAd Easy system. MIN6 cells were transfected with AdGFP and AdWdr13 with 100 MOI each in 24 well plates. After 48 hours of transfection, overexpression of Wdr13 gene was confirmed by western blot (Figure 5A) using anti WDR13 antibody. Further, we studied the growth curve of MIN6 cells by counting cells after overexpression of Wdr13 gene. A significant reduction in cell growth was observed 48 h post transfection (Figure 5A). Effect of WDR13 on cell cycle regulators To understand the growth retardation after overexpression of WDR13, various cell cycle regulators were analyzed by western blotting. The protein levels of cyclin D1, cyclin D2, cyclin E and p27 were unaltered, whereas p21 was highly upregulated (Figure 5B). Conversely, siRNA knockdown of Wdr13 gene showed downregulation of p21 protein levels (Figure 5C). Further, consistent with these results western blot analysis revealed reduction in p21 levels in the islets from Wdr13 knockout mice, (Figure 5D). To understand the nature of p21 regulation by WDR13, promoter occupancy of WDR13 protein was analyzed at p21 promoter. Chromatin immunoprecipitation experiment showed interaction of WDR13 with p21 promoter sequences indicating a role of WDR13 in p21 regulation (Figure 5E). Discussion In the present study, we have examined the in vivo role of one of the WD repeat proteins, namely, WDR13 by knocking out this gene in mouse. The mutant mice were viable and fertile without any overt phenotype except that the mice were significantly heavier than their wild type littermates at around nine months and continued to weigh more till the termination of the growth experiment at 12 months when fed on normal chow. This age- PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38685