Conditional inactivation of Slc11a2 in the intestine. Slc11a2 wild-type locus (A) and targeted locus (B) after introduction of an intronic floxed neomycin resistance and cytosine deaminase cassettes and an additional loxP site between exons 5 and 6. Positions of 5′ and 3′ probes used for Southern blot analysis are shown as red bars labeled A and B, respectively. (C) Partial Cre recombinase–mediated excision in which the Neo and cytosine deaminase cassettes were removed but the coding sequence was left intact. (D) Slc11a2 locus after complete excision to inactivate the gene. (E) Southern blot analysis of the 5′ end of the locus in ES cells after Cre transfection. (F) Southern blot analysis of the 3′ end of the locus in ES cells after Cre transfection. Clone 3 retained the floxed allele and was used to generate Slc11a2^flox/flox mice; clones 2, 5, and 6 underwent complete deletion and were used to generate Slc11a2^–/– mice without residual Neo and cytosine deaminase cassettes. (G) Immunoblot analysis of duodenum from Slc11a2^–/– (lane 1), Slc11a2^int/int (lane 2), wild-type (lane 3) and Trf^hpx/hpx (lane 4) mice. Protein amounts loaded per gel were 4 μg of Slc11a2^–/–, 2 μg of Slc11a2^int/int, 50 μg of wild-type, and 2 μg of Trf^hpx/hpx duodenal lysates. Slc11a2 migrates as a diffuse band between the 60-kDa and 85-kDa markers (arrow). (H) Southern blot analysis of Slc11a2^int/int mice resulting from an intercross of Slc11a2^flox/flox and Villin-Cre mice using probe A, demonstrating that the deletion of the floxed region occurred only in intestinal tissues.