Generation of Flvcr mutant mice. Using cDNA sequences for feline and human FLVCR, we identified the cDNA sequence of murine Flvcr from EST data in the Genbank database, and confirmed this by amplification and sequencing of exons 1, 5, and 8 from genomic DNA. We next identified BAC contigs containing the entire 18 kb murine Flvcr genomic region, confirming it was the Flvcr locus and not a paralog (S1). We then derived the intron/exon structure by aligning the cDNA and genomic sequences. Due to

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... e presence of a gene on the opposite strand that likely shares a promoter region with Flvcr (S1), we opted to delete exon 3, which encodes the intracellular loop between transmembrane domains 6 and 7, as our knockout strategy. Structure/function analyses of two proteins structurally related to FLVCR, the lactose permease and the reduced folate carrier protein, revealed that this loop is required for protein stability, processing, and function in prokaryotic and eukaryotic cells (S2-4). Exon 3 deletion should, therefore, produce an unstable protein that cannot traffic to the membrane (S2, S3). If (unexpectedly) the protein were to successfully transport to the membrane, its structure is predicted to be severely altered, making it unlikely to retain function (S5). The construct consists of a loxP site in the intron between exon 2 and exon 3, and a PGK promoter-driven neomycin selection cassette, flanked with loxP sites in the intron between exon 3 and exon 4 (Fig. S1 ). Primers were designed for a PCR-based cloning strategy and we generated the targeting construct using template genomic DNA that matched the strain of the ES cells (129S4/SvJae). All exons and intron-exon boundaries were sequenced in the construct and matched sequences from a BAC clone. The ES culture, transfection, cloning, and screening were performed as before (S6). Targeted clones were transfected with a cre expression vector and recloned to identify flox and null allele containing clones by Southern blot and PCR analysis. A flox containing ES clone with a normal karyotype was used for blastocyst injection and the male chimeras were bred to female C57BL/6 mice. Heterozygous Flvcr +/flox mice were bred to either Cmv-cre + or Mx-cre + transgenic mice (S7, S8) to generate Flvcr +/-;Cmv-cre + or Flvcr +/flox ;Mxcre + mice, respectively. Mice were backcrossed to C57BL/6 for 1 to 5 generations then intercrossed to obtain F1, F2, and F3 generations for use in these studies. We then confirmed that exon 3 deletion produced a nonfunctional protein by cloning the deleted allele and appropriate controls into NRK cells which normally do not express FLVCR and performed export studies using zinc mesoporphyrin which is a fluorescent heme analogue that is transported comparably to heme (Fig. S2 ). Cells expressing FLVCR show export at 90 minutes. Cells expressing the exon 3 mutant fail to export ZnMP, and cells expressing both wild-type and exon 3-deleted FLVCR show heme export. This latter findings, proves that the mutated protein does not act as a dominant-negative protein. Futhermore, the lack of a dominant-negative effect suggests that FLVCR functions as a monomer, which is consistent with the known crystal structure of MFS members (S4, S9). Breeding and analysis of mutant Flvcr mice. Animals were housed in a SPF facility at the University of Washington, Seattle. The Institutional Animal Care and Use Committee approved all studies. Embryos were obtained from timed pregnant females for genotyping, immunohistochemistry, or pathologic analysis. Embryos were staged according to standard methods; noon of the day when a copulatory plug was identified is 0.5 dpc. To induce Mx-cre