Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein
J. Clin. Invest. Fayez F. Safadi, et al. 103:239 doi:10.1172/JCI5244 [Go to this article.]

Figure 1
Targeted disruption of the mouse DBP locus. (a) A fragment of mouse genomic DNA containing exons 4–8 of the DBP gene was used to design the targeting vector. A PGK-promoter/neomycin phosphotransferase cassette was inserted at the BamHI (M) site in exon 5 to disrupt the mDBP gene and provide for positive selection. A DTA cassette was ligated to the 5′ HindIII site for selection against random integration. (b) Restriction enzyme mapping distinguished the intact from the disrupted DBP allele. A mouse DBP exon 2 probe, located outside of the targeting vector itself, hybridized to an 8.8-kb EcoRI (R) fragment from the native DBP allele and a 5.7-kb EcoRI fragment from the disrupted mDBP allele. Restriction sites are: S, SalI; H, HindIII; B, BglII; R, EcoRI; C, ClaI; M, BamHI. (c) The targeting vector (a) was transfected into ES cells, G418 selection was applied, and surviving clones were analyzed by Southern blotting. Analysis of 8 representative ES cell lines among the 65 examined is shown. All eight clones contained the native 8.8-kb mouse DBP EcoRI fragment, and one clone (D1) also contained the 5.7-kb fragment, indicative of successful homologous recombination. DBP, vitamin D binding protein; DTA, diphtheria toxin A; ES, embryonic stem.