Fto and Postnatal Growth Figure 8. The body composition of Fto neural-specific knockout mice. (A) Fat mass of 16-week-old FtoN+ and FtoND mice. P = 0.4551(male), 0.1681(female). (B) Lean mass of 16-week-old FtoN+ and FtoND mice. (C) Total tissue mass of 16-week-old FtoN+ and FtoND mice. P,0.0001(male), and P = 0.0175(female). (D) Body composition (fat mass/total tissue mass %) of 16-week-old FtoN+ and FtoND mice. P = 0.0118(male), 0.0024(female). In (A)– (D), for each genotype (FtoN+/FtoND), n = 19/21(male), 19/17(female). Statistical analyses were performed by unpaired t-test. *P,0.05, **P,0.01. All values are mean 6 s.e.m. doi:10.1371/journal.pone.0014005.g008 growth. However, whether FTO plays a role in obesity needs further investigation. The reduced serum IGF-1 levels and a significant decrease in bone mineral density in both whole body and neural Fto knockout models argue for a function of Fto in the hypothalamus-pituitary axis. This axis controls the expression and secretion of IGF-1 by the liver through the action of growth hormone (GH), and produces a host of other endocrine factors involved in the regulation of many aspects of normal physiology including mineral metabolism in the bone [45,46]. GH deficiency is associated with obesity in humans [47] and the loss of GH function due to a missense mutation in mice results in disproportional increases in body fat despite an overall reduction in body size and weight [48]. It is possible that the Fto mutant mice suffer some degrees of GH deficiency. Determining whether or not FTO regulates GH and/ or other hormones secreted by the hypothalamus-pituitary axis will greatly facilitate the elucidation of FTO’s physiological function in future. Our neural-specific knockout mice recapitulated essentially all of the phenotypes in the complete knockout mice. This indicates that FTO function in the brain is crucial in spite of its ubiquitous expression. The analysis of mice with sub-regional deletion of Fto in the brain, especially, the hypothalamus-specific deletion, will pinpoint where exactly Fto functions. As a member of the Fe (II) and 2-oxoglutarate (2-OG)-dependent oxygenase superfamily [25–27], FTO belongs to the AlkB subfamily [25–27,30]. AlkB proteins are important enzymes catalyzing the removal of alkyl adducts from DNA in both prokaryote and eukaryote organisms [49]. More recent biochemical analysis suggested that FTO might be a RNA demethylase [30], and recent structure analysis of FTO also supported its preference for single- stranded over double-stranded nucleic acids [25]. This is more consistent with a role of FTO in regualting gene expression (at the posttranscriptional level) than in reparing DNA. Likewise, a role of FTO in the regulation of gene expression is more fitting with the phenotypes of Fto mutnat mice than a role in DNA repair. Knowing where exactly FTO funcitons will assist greatly in the identification of its physiological substrates. The conditonal Fto knockout mice we have generated will be helpful in that regard. Materials and Methods Ethics Statement All animal experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine. The Protocol number is AN-5002. Western blot analysis and antibodies For Western blot analysis, tissues were lysed in RIPA buffer and the protein concentration was determined with Bradford method. We raised rabbit anti-mFTO antisera and affinity-purified the antibodies for the use in immunoblotting. Anti-a-TUBULIN antibodies were obtained from Sigma (T 5168). Generation of Fto conditional knockout mice The genomic DNA of targeting mouse Fto sequence containing exon 3 (about 9 kb) was isolated from a BAC clone (Sanger Center, UK) by gap repair. The construction of the conditional targeting vector was carried out via homologous recombination in E. coli [50]. The vector was linearized and introduced into E14Tg2a.4 ES cells. Recombinant ES clones were identified by Southern blot analysis and used to produce chimeric mice. The F1 Fto conditional knockout mice (Fto+/cko) were bred with Meox2-Cre mice (JAX 003755) expressing Cre recombinase in all the epiblast-derived tissues to delete exon3. The progenies were PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e14005