Generation of Hfe^VillinCre mice. (A) Duodenum-specific Hfe knock-out mice were generated by crossing mice carrying a floxed Hfe allele (Hfe^flox) with mice expressing the Cre recombinase under the control of the murine villin promoter. Cre-mediated excision of exons 3 to 5 generates an Hfe null allele. PCR primers F and R1 amplify a 646-bp (base pair) fragment from the intact Hfe^flox allele, whereas primers F and R2 amplify a 796-bp fragment from the recombined allele. Under the PCR conditions used, these primers fail to amplify the predicted 2827-bp fragment from the full-length Hfe^flox allele. Exons are symbolized by numbered boxes, triangles indicate the loxP sites. (B) The Hfe^flox allele is recombined specifically in the intestine. Recombination of the Hfe^flox allele along the intestinal axis in Hfe^VillinCre mice expressing the Cre recombinase (Cre⁺) and in control littermates (Cre⁻) is monitored by PCR analysis using the primers detailed in Figure 1A. The numbers above each lane refer to the corresponding intestinal sections: 1, 0 to 2 cm; 2, 2 to 4 cm; 3, 4 to 6 cm; 4, 6 to 8 cm; 5, 8 to 10 cm; 6, 10 to 12 cm downstream of the pylorus. (C) Tissue specificity of Hfe recombination was assessed by diagnostic PCR analysis. The duodenum samples correspond to the proximal 2 cm of the intestine. Hfe^−/− and Hfe^flox (Cre−) samples were used as positive and negative controls, respectively.