filled follicles of the anterior pituitary containing PAS (periodic- acid Schiff reaction) positive material have been reported in several vertebrates including humans [5,6]. These follicles have been known to increase in number and size with age. The content of the follicles was reported to include polysaccharides and glycoproteins but none of the known pituitary hormones have hitherto been localized to them. To our knowledge, the function and significance of these follicles is still unknown. Here we generated mutant mice with a novel targeted Cthrc1 allele and focused on the analysis of their phenotype in adulthood. Monoclonal antibodies were generated against C terminal and N terminal epitopes of Cthrc1, which allowed us to localize Cthrc1 in tissues of a variety of species including pig. Our results demonstrate circulating levels of Cthrc1 in human plasma including expression in the anterior pituitary as well as neurose- cretory nuclei of the hypothalamus. Materials and Methods All protocols involving animals were approved by the In- stitutional Animal Care and Use Committee of the Maine Medical Center (protocol numbers 0905 and 1112) and were in compliance with all applicable regulations and guidelines including the National Institutes of Health Guide for Care and Use of Laboratory Animals. All surgical interventions were performed under general anesthesia with tribromoethanol/tert. amyl alcohol. Human plasma samples were obtained under a protocol approved by the Institutional Review Board of the Maine Medical Center (protocol number 3657). Animals We generated a novel Cthrc1 allele by replacing exons 2, 3 and 4 with a neomycin cassette using the targeting vector pKO Scrambler NTKV-1905 (Stratagene) (Fig. 1A). Exon 1 contains only 59 untranslated sequence and the N-terminal 52 amino acids, which include the signal sequence plus additional 20 amino acids. Embryonic stem cell clones were screened by Southern blotting and positive clones injected into blastocysts (Fig. 1B). Two chimeras were obtained and bred with C57BL/6J mice (Jackson Laboratory), producing offspring with agouti coat color indicating germ line transmission. Their genotypes were verified by Southern blotting and a PCR screen that amplified the wild-type allele (primers: 59-CCACTGGAAACCTCTGGAGTTG-39 and 59- AAGTTCACACAAAGGAAGCCCCGC-39) and the mutant allele (primers: 59-GTGTGTTTTGAGGTGTGGTCCC-39 and 59TGGATGTGGAATGTGTGCGAGG-3). These animals have been backcrossed on the 129S6/SvEv (Taconic) or C57BL/6J background for 11 generations. Cthrc1 mice for experiments were derived from matings of homozygous mutants. The mutant allele was backcrossed regularly with 129S6/SvEv or C57BL/6J breeder stock to prevent genetic drift. The age of the mice used for specific experiments is indicated in the figure legends. Mice were fed a standard rodent diet (Harlan, 2018 Teklad Global 18% Protein Rodent Diet) and water ad libitum, and housed with dry cellulose bedding (Harlan, 7070 Diamond) under a 14 hour daylight-10 hour night cycle. Litter size was determined as the number of pups weaned at the age of 21 days after birth. Growth curves were obtained for Cthrc1 and wild type mice on the 129S6/SvEv (n = 5–33 per time point). Livers of seven month old mixed gender Cthrc1 and wild type mice on the C57Bl/6J background were examined to assess histology and lipid content. Lipid storage was determined by image analysis of oil red O stained sections using the same approach as described previously [4]. Hormonal Functions of Cthrc1 Pig tissues were obtained from euthanized animals (6 to 12 months of age) of the Advanced Trauma Operative Management (ATOM) courses conducted at our facility or from a local slaughterhouse. Primer sequences used for RT-PCR of pig cDNA were 59- GACCCCTTCATTGACCTCCACTAC-39 and 59- ACATACTCAGCACCAGCATCGC-39 for Gapdh and 59- GAATGCCTGAGGGAAATCTTTGAG-39 and 59- CGTCTCCTTTTGGGTAATCTGCG-39 for Cthrc1. The an- nealing temperature was 55.9uC and 35 cycles of amplification were performed. Rat tissues from three month old male Sprague Dawley rats were kindly provided by Dr. Rene´e LeClair (University of New England, Biddeford, ME). Glycogen Assay Mixed gender wild type and Cthrc1 mice on the 129S6/ SvEv background were used for this assay (n = 4–23 animals per group, 3–5 months of age). Samples were obtained from identical sites of the liver and the right gastrocnemius muscle. Glycogen from 10 mg fresh-frozen tissue was hydrolyzed to glucose in 500 ml of 2 N HCl for 2 h at 100uC with vortexing every 30 minutes. Samples were neutralized with 500 ml 2 N NaOH and 50 ml 1 M TRIS pH = 7.6. The glucose hexokinase reagent (ThermoFisher) was used as directed. A standard curve was established with glucose standards covering the range of 0.00312 to 0.1 mmole. Using GraphPad Prism software, concentrations of glucose in the samples were calculated based on the standard curve. Three different samples from each organ were assayed in duplicates and mean values were calculated and compared using Student’s t-test. The assay was performed four times with separate sets of samples from different mice and similar results were obtained each time. The results of a representative experiment are shown. Body Fat and Blood Triglyceride Levels Body composition was determined as described using a Lunar PIXImus II Mouse Densitometer (GE Medical Systems) [7]. Blood triglyceride levels were determined in non-fasted 3–4 month old mixed gender wild type and mutant mice on the 129S6/SvEv background using test strips for the One Touch Ultra system. Glucose Tolerance and Insulin Stress Test For glucose tolerance testing, mixed gender wild type and Cthrc1 mice on the 129S6/SvEv background (n = 8, 3–4 months of age) were injected intraperitoneally with 1 g of glucose per kg of body weight after 16 hours of fasting. For insulin stress testing, mixed gender, non-fasted wild type and Cthrc1 mice on the 129S6/SvEv background (n = 8, 3–4 months of age) were injected intraperitoneally with insulin (1 U/kg, NovoLog, Novo Nordisk). Blood was obtained for glucose level measurements using the One Touch Ultra glucometer at the indicated times. Rabbit Monoclonal Antibody Generation, Immunohistochemistry, and Immunoblotting A rabbit monoclonal antibody was raised against a synthetic peptide of the conserved C terminus of Cthrc1 (GDASTGWNSVSRIIIEELP) using the services of Epitomics, Inc. (Burlingame, CA). Clone Vli-55 was suitable for Western blotting and immunohistochemistry on paraffin-embedded, para- formaldehyde-fixed tissues. Rabbit monoclonal Vli-55 was used at 20 ng/ml for immunohistochemistry on paraffin-embedded, formalin-fixed tissue section following antigen retrieval with citrate buffer (0.1 M, pH = 6.0). Subsequent steps of the immunostaining PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47142