Materials and Methods Ethics statement The institutional Animal Ethics Committee of the Centre for Cellular and Molecular Biology, Hyderabad, India approved all the mice experiments. Generation of Wdr13 mice To construct the Wdr13 gene targeting vector, a 7.1 kb HindIII fragment from this gene including exon1 to exon7 was sub-cloned in pBluescript II KS vector (Stratagene). A 1.35 kb region from this fragment spanning exon 2 and exon 3 (partial) was replaced by XhoI-SalI fragment of pMC1neo Poly A (Stratagene). To further enrich for the targeting events, a negatively selectable HSV-tk gene was placed before the 59 end of the homologous sequences from Wdr13 gene. The resulting vector had homologies of 1.6 kb and 4.1 kb at the 59 and 39ends, respectively. Forty micrograms of linearized targeting vector DNA was electroporated into R1 ES cells [46]. The ES cells were selected with G418 (0.25 mg/ml) and ganciclovir (2 mM). To identify the targeted clones, genomic DNA was isolated from ES cells and southern hybridization was performed using a 700 bp EcoRV-BamHI fragment as a probe from the 59 end of this locus. One of the targeted clones was injected into 3.5-dpc C57BL/6 blastocysts and the latter were transferred into the uteri of CD1 pseudopregnant females. To obtain germline transmission of the mutant allele, chimaeric male mice were mated with CD1 females. Germ line transmission was confirmed by southern analysis. PCR was used for regular screening of mutant mice using a primer pair (Table S3) from the Wdr13 locus and another primer pair from neomycin gene. Northern blot and western blot analysis Various tissues were snap frozen in liquid nitrogen and stored at 280uC till further use. Total RNA was isolated using RNeasy Mini Kit (Qiagen). For Northern analysis, RNA was electrophor- esed on 1% agarose gel containing 2.2 M formaldehyde and blotted on hybond N+ membrane with 50 mM NaOH. Wdr13 cDNA was radio labeled with adATP using random priming kit. Hybridization was performed overnight at 65uC in 0.5 M phosphate buffer/7% SDS/1 mM EDTA and the membranes were then washed 36 at 65uC in 40 mM phosphate buffer/1% SDS/1 mM EDTA. The membranes were exposed to X-ray sheets (Fuji films) and developed. Proteins were extracted from various tissues, separated on 10% SDS-PAGE, blotted on PVDF membranes and western blots were performed. Anti-WDR13 antibody (HPA000913) from Sigma and p21 (sc-397), p27 (sc-528), cyclinD1 (sc-246), cyclinD2 (sc-593) and cyclinE (sc-481) from Santacruz were used for visualization of the respective protein. Animal weight measurement, feed consumption and body composition Mice were housed in temperature, humidity and light/dark cycle (12 hrs 6am- 6pm) controlled animal rooms. Autoclaved normal diet or high fat diet was fed ad libitum, and the feed intake was measured weekly. Mice were weighed fortnightly. To determine the body composition, mice were dissected and the weights of various organs were measured. GTT, ITT, and insulin measurements Blood glucose was measured with a glucometer using Sensor Comfort strips (Accu-Check) and insulin was measured by ELISA kits (Linco Research). To compare glucose clearance and insulin secretion, the mice were kept off-feed for 16 hours, glucose was Role of Wdr13 Gene in Beta Cell Proliferation injected intraperitoneally (2.0 grams glucose/Kg body weight) and blood was collected at 0, 30, 60 and 120 minutes intervals. Insulin tolerance was estimated by measuring glucose levels at 0, 30, 60 and 120 minutes intervals after injecting human insulin intraper- itoneally (1 U/kg). Islet isolation and in-vitro insulin secretion Pancreatic islets were isolated from the wild type and knockout mice as described by Wen et al. [26]. Briefly, 2 mg/ml collagenase type IV (Invitrogen) dissolved in Hanks’ balanced salt solution (HBSS) was injected into common bile duct and pancreas were incubated at 37uC for 30 minutes. After collagenase digestion, pancreatic tissue was washed twice with cold HBSS and islets were purified by Ficoll gradient. The purified islets were washed two times with HBSS and transferred to RPMI1640 medium supplemented with 10% fetal bovine serum 100 U/ml penicillin, and 50 mg/ml streptomycin for overnight recovery. The islets were transferred next morning to Krebs–Ringer bicarbonate buffer (111 mM NaCl, 4.8 mM KCl, 2.3 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3) supplemented with 10 mM HEES, 2.8 mM glucose, 0.2% BSA and incubated for 90 minutes at 37uC with 5% CO2. To measure the insulin secretion in vitro, five equal size islets were plated (in triplicate) in a single well of 96-well plate containing either 250 ml Krebs–Ringer bicarbonate buffer/ 2.8 mM glucose or 250 ml Krebs–Ringer bicarbonate buffer/ 16.0 mM glucose and were incubated for 1 h at 37uC with 5% CO2. The supernatant was collected and stored at 280uC for insulin measurement. Cell proliferation assay, histology and immunostaining To assay beta cell proliferation, BrdU was given to mice in drinking water (1 mg/ml) for 7 days. For histological examination, tissues were fixed overnight in buffered 4% para formaldehyde, embedded in paraffin and sectioned (4 mm thickness). Sections were mounted on positively charged slides (Fisher Scientific) and were stained with either hematoxylin-eosin or subjected to immunostaining with anti-insulin (R&D System) and anti-BrdU (BD Biosciences) antibodies. Primary antibody was detected by cy3 or FITC-conjugated antibodies. Pancreatic and islet areas were measured from pancreatic sections from four mice each of the wild type and mutant genotypes using Axioskop (Axivision software). Islet mass per pancreas was calculated by multiplying relative islet area with wet mass of pancreas. Adenovirus generation and cell proliferation assay To overexpress WDR13 protein in various cell lines, Wdr13 adenovirus constructs were generated. In brief, Wdr13 cDNA was amplified from pCMV-FLAG-Wdr13 vector using T7 forward primer and reverse primer 59GCTCTAGAGCAGCACAGGGT- GACAGAACC39, digested with PmeI and cloned at EcoRV of pAdTrack-CMV vector. AdGFP or AdWdr13 were generated in HEK293T packaging cell line according to He et al., [47]. 10,000 MIN6 cells (obtained from National Centre for Cell Science, Pune, India) were seeded per well of 24 well plates [48] in DMEM media containing 10% FBS and were transfected either with AdGFP or AdWdr13 using a titer of 100 MOI. Cell number was monitored by MTT assay at 24 h interval. For western blot analysis, MIN6 cells were transfected with AdGFP or AdWdr13 virus (100 MOI) and the cells were lysed 48 hour post transfection. PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38685