Generating Intestine-Specific Bpnt1 Knockout Mice

Bpnt1 floxed mice were generated using a standard homologous recombination approach (Fig. S2A). Briefly, we recombined the fourth and fifth exons of the Bpnt1 locus in 129/SvEv ES cells with a construct containing flanking LoxP sites and a neomycin resistance cassette. Cells resistant to neomycin were confirmed by PCR and Southern blotting, injected into blastocysts, and implanted into pseudopregnant females by the University of North Carolina Animal Models Core. Chimeric founders were identified by Southern blotting and PCR and crossed with B6.Cg-Tg(ACTFLPe)9205Dym/J mice (The Jackson Laboratory) expressing FLP recombinase to remove the neomycin cassette. Bpnt1^+/fl mice were then backcrossed four generations into the C57BL/6J background, intercrossed, and maintained as Bpnt1^fl/fl animals. To obtain intestine-specific knockouts, we first crossed B6.SJL-Tg(Vil-cre)997Gum/J mice (The Jackson Laboratory) expressing Cre recombinase under the control of the villin promoter with Bpnt1^+/− animals to obtain Bpnt1^+/−Vil-Cre⁺ double heterozygotes. These animals were then crossed to Bpnt1^fl/fl mice to generate Bpnt1^+/fl, Bpnt1^−/fl, Bpnt1^+/int, and Bpnt1^−/int mice. Wild-type and conventional knockout Bpnt1 alleles were genotyped by multiplex PCR using the following primers: (i) 5′-cctatagtcctagcacttgagagg-3′, (ii) 5′-accaaagaacggagccggttggcg-3′, and (iii) 5′-aggtcggaaccctgttctctagtc-3′. Floxed Bpnt1 alleles were genotyped by PCR using the following primers: (i) 5′-cttgtggtttgggttgaccccttag-3′ and (ii) 5′-ctctagcccagtcagacatgtcag-3′. Villin-Cre expression was determined by PCR using the following primers: (i) 5′-gcggtctggcagtaaaaactatc-3′ and (ii) 5′-gtgaaacagcattgctgtcactt-3′. All animals unless otherwise noted were maintained on Purina 5058 natural products chow. Animals receiving supplemental iron were injected into the scruff of the neck weekly for a total of 3 wk with 5 mg of sterile Fe–dextran (Sigma). After 3 wk, mice were killed and analyzed as described above. Animals challenged with iron-deficient diets were maintained on normal Purina 5058 chow until the time of weaning (P23), at which point they were given either iron-deficient AIN-93G–defined chow with 2–6 ppm total iron or an identical AIN-93G supplemented with 200 ppm iron(II) sulfate (Harlan-Teklad TD.120105 and TD.120106, respectively). Mice were killed after 5 wk of dietary treatment and analyzed for hematological parameters as described above. Animal care and experiments were performed in accordance with the National Institutes of Health guidelines and approved by the Duke University Institutional Animal Care and Use Committee.