Fto and Postnatal Growth Figure 9. The metabolic parameters of Fto neural-specific knockout mice. (A) Average hourly oxygen consumption of 16,17-week-old male FtoN+ and FtoND mice during the light and dark period. P = 0.0105 (light), and P = 0.0044 (dark). (B) Average hourly oxygen consumption divided by lean mass. P = 0.00556 (light), and P = 0.0233(dark). (C) Average hourly carbon dioxide production of 16,17-week-old male FtoN+ and FtoND mice during the light and dark period. P = 0.004 (light), and P = 0.0196(dark). (D) Average hourly carbon dioxide production divided by lean mass. P = 0.0131(light), and P = 0.0142(dark). (E) Accumulative food intake over a period of 72 hours of 16,17-week-old male FtoN+ and FtoND mice. P = 0.4601. (F) Food intake divided by lean mass. In (A)–(F), the number of animals used were 7 (FtoN+) and 9 (FtoND). 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.g009 backcrossed to C57BL/6 mice for at least two more generations. The heterozygotes (F3 and beyond) were then intercrossed to generate complete knockout mice. F1 Fto+/cko mice were also bred with mice expressing FLP1 recombinase (JAX 003946) to first delete the selection cassette flanked by the Frt sites. The mice with one floxed Fto allele (Fto+/flox) were then bred with Meox2-Cre mice (JAX 003755) to delete exon 3. Similarly, the progenies with one Fto knockout allele (having no selection cassette now) were backcrossed to C57BL/6 mice for at least three more generations. The heterozygotes were then intercrossed to generate knockout mice. To generate brain specific Fto deletion, Ftoflox/flox mice were crossed with Nestin-Cre mice (JAX 003771) to generate Fto+/flox/Tg(Nes-Cre) mice, which were then crossed to Ftoflox/flox mice to generate FtoN+ (Ftoflox/flox and Fto+/flox, for control) and FtoND (Ftoflox/flox/Nes-Cre) mice. Animal experiments Animals were housed in a specific pathogen free facility at 2262uC under a cycle of 12 hr light (7:00 am light on) and 12 hr dark (7:00 pm light off). They have free access to water and food (normal chow: 2920X Teklad Rodent Diet). For 24 hr fasting experiments, the food was removed from the cages at 6:00 pm, but PLoS ONE | www.plosone.org 11 November 2010 | Volume 5 | Issue 11 | e14005