Generation of <i>fgf22</i> knockout mice.

<p>A) Targeting strategy for the <i>fgf22</i> knockout mouse. Schematic representations of the endogenous mouse <i>fgf22</i> locus (middle), targeting construct (top) and disrupted allele (bottom). Black boxes in the wild type allele represent exons 1 to 3. E indicates <i>EcoRI</i> restriction enzyme recognition sites. PCR primers used for genotyping are represented by arrows. B) Southern blots showing successful recombination in ES cells used for generation of <i>fgf22</i> null mice. Of 192 ES clones analysed, two scored positive for homologous recombination (1C12 and 1E1) and displayed identical hybridisation patterns. C) PCR analysis of genomic DNA isolated from ear snips of an heterozygous breeding pair of both identified mutant clones. <i>Fgf22</i> wt (+/+) samples show a single band at 286 bp (mice 1, 3 and 4), <i>fgf22</i> ko (−/−) display a single ko allele at 130 bp (mice 2 and 7) and heterozygous (+/−) samples amplify both wt and ko alleles (mice 5, 6 and 8). D) Confirmation of gene deletion. RT-PCR analysis of cDNA generated from mouse brain. Samples from wild type mice (+/+) display an intense <i>fgf22</i> band, <i>fgf22</i> heterozygous samples (+/−) display same size band of decreased intensity and <i>fgf22</i> knockout samples (−/−) lack the presence of a correct size band. GAPDH primers were used as a control for RNA quality and concentration. Blank represents PCR reaction mix without cDNA.