Fto and Postnatal Growth Generation of Fto deletion mice In order to investigate the physiological function of Fto in mice, we generated a conditional knockout (cko) line by flanking exon 3 with two loxP sites (Figure 2A). Exon 3 encodes about 40% of the protein. Deletion of exon 3 would result in frame-shift of the downstream exons and early termination in translation (Figure 2B). Germline transmission of the cko allele (Ftocko) was obtained and confirmed by Southern blot analysis (Figure 2C). F1 Fto+/cko mice were either directly bred to Meox2-Cre [34] mice to generate a knockout allele (Fto*D) still bearing the neo selection cassette, or first bred to Flpase-expressing mice [35] to generate a floxed allele (Ftoflox). Fto+/flox mice were then crossed to Meox2-Cre mice to generate the clean deletion of exon 3 allele (Fto#D) (Figure 2A). Both Fto#D and Fto*D were used as knockout in this study and will be referred together as FtoD from hereon, since there is no phenotypic difference between Fto#D/#D and Fto*D/*D mice. Homozygous knockout mice (FtoD/D) were obtained by crossing of heterozygous pairs, and the absence of FTO protein expression in FtoD/D mice was confirmed by Western blot analysis (Figure 2D). Complete depletion of Fto in mice results in postnatal growth retardation FtoD/D mice are viable, but only about 50% of them could survive to postnatal day 10–14 (Table S1) as has been reported in Figure 2. Generation of Fto knockout mice. (A) Strategy to generate various Fto alleles. Hind III indicates the enzyme cutting sites for Southern blot analysis in (C). (B) Schematic representation of predicted Fto wildtype and knockout coding sequences (CDS). (C) Southern blot analysis of wildtype and cko alleles. The genomic DNA was digested by Hind III, separated on agarose gel, blotted, and hybridized with the indicated probes as shown in (A). (D) Western blot analysis of different tissues isolated from wildtype (+/+), heterozygous (+/D) and homozygous (D/D) mice. doi:10.1371/journal.pone.0014005.g002 PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e14005