Generation of a conditional Fgf23 allele

Exon 2 of the mouse Fgf23 gene was targeted for Cre-mediated recombination, since deletion of this exon is predicted to result in a truncation of FGF23 protein (Supplemental Figure 1). To this end, a 9.4 kb target region of mouse Fgf23 identified from BAC clone RP23:142M1 was subcloned into the pSP72 backbone plasmid. A single LoxP site was inserted downstream of exon 2. The 1.7 kb pGK-gb2 LoxP/FRT-flanking Neomycin cassette (Neo^R) was inserted upstream of exon 2, which contained new BamHI and NcoI restriction sites. Restriction enzyme digests and sequencing confirmed the final targeting vector (depicted in Supplemental Figure 2). The targeting vector was separated from the vector backbone with NotI and injected into BA1 ES cells. Positive ES cell clones were identified with G418 selection, and homologous recombination events within the Fgf23 locus (Supplemental Figure 2) were confirmed with PCR and Southern blotting (Supplemental Figure 2). ES cell DNA were digested with NcoI and probed with a 462 bp 3′-external probe for long arm recombination. DNA digested with BamHI and probed with a 515 bp 5′- internal probe confirmed short arm homologous recombination (Supplemental Figure 2) as designed by InGenious, Inc. (Ronkonkoma, NY).