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. 2011 May 19;117(20):5494-502.
doi: 10.1182/blood-2010-11-319483. Epub 2011 Feb 10.

Targeted deletion of the mouse Mitoferrin1 gene: from anemia to protoporphyria

Marie-Berengere Troadec  1 , David Warner, Jared Wallace, Kirk Thomas, Gerald J Spangrude, John Phillips, Oleh Khalimonchuk, Barry H Paw, Diane McVey Ward, Jerry Kaplan
Affiliations

Targeted deletion of the mouse Mitoferrin1 gene: from anemia to protoporphyria

VSports手机版 - Marie-Berengere Troadec et al. Blood. .

Abstract

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions VSports手机版. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show that the activity of mitoferrin1 is required to manage an increase in heme synthesis. The data also show that alterations in heme synthesis within hepatocytes can lead to protoporphyria and hepatotoxicity. .

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Figures

Figure 1
Figure 1
Generation of floxed Mfrn1 alleles. Schematic description of the wild-type Mfrn1 locus (A) and the locus after introduction of a loxP-FRT-neomycin resistance-FRT cassette in intron 1 and an additional loxP site between exons 2 and 3 (B). The position of the 5′-flanking probe used for Southern blot analysis is shown as a blue bar in panels A and B. (C) Flp recombinase–mediated excision in which the neomycin cassette was removed by FRT recombination but the coding sequence was left intact. (D) Complete Cre recombinase–mediated excision of exon 2 to inactivate the Mfrn1 gene. For panels A through D, LoxP sites are shown as red triangles and FRT sites are shown as green diamonds. (E) Southern blot analysis of the 5′ end of the locus in ES cells after BamHI digestion. Lane 1 shows the wild-type allele at 7.4 kb, and lanes 2-4 targeted recombination with a wild-type allele at 7.4 kb and a recombinant allele at 4.5 kb, as indicated in panels A and B. (F) PCR analysis of the offspring of Mfrn1floxneo/+ and Mfrn1+/+ chimeric mice. PCR was done using primers F, R1, and R2, as indicated in panels A and B. Lane 1 and lane 4 show the Mfrn1floxneo/+ genotype and lanes 2 and 3 the Mfrn1+/+ genotype. The presence of Neo (primers F × R1) gives a product of 120 bp; the absence of Neo (primers F × R2) gives a product of 60 bp. (G) PCR analysis of Cre recombinase–mediated excision of exon 2. PCR was done using primers F, R3, and R4, as indicated in panels C and D. The wild-type allele gives a product of 173 bp (primers F × R3), the flox allele gives a product of 273 bp (primers F × R3), the negative allele gives a product of 428 bp (primers F × R4), and R3 does not hybridize anymore. Lane 1 shows the Mfrn1flox/− genotype, lane 2 shows the Mfrn1+/− genotype, lanes 3 and 4 show the Mfrn1flox/+ genotype, and lane 5 shows the Mfrn1+/+ genotype.
Figure 2
Figure 2
Embryonic lethality of a homozygous Mfrn1 deletion. (A) PCR analysis of the offspring from the breeding of Mfrn1+/− with Mfrn1+/− mice. These littermates were genotyped at E9.5. In this particular litter, 1 animal was wild-type, 6 were heterozygous, and 1 was homozygous for the Mfrn1 deletion. PCR was done using primers F, R3, and R4, as indicated in the legend to Figure 1. (B) Western blot of whole embryos probed with antibodies against Mfrn1, Mfrn2, and tubulin using peroxidase-conjugated goat anti–rabbit IgG or goat anti–mouse IgG. (C-H) Stereomicroscopy of E9.5 embryos from Mfrn1+/− × Mfrn1+/− breeding. Panels C, E, and G are the Mfrn1+/− mouse; panels D, F, and H are the Mfrn1−/− mouse. Panels C and D show embryos kept in the yolk sac, panels E and F show embryos without their yolk sac, and panels G and H are enlarged images from panels E and F. The Mfrn1−/− mouse is deprived of hemoglobin, as shown by the lack of RBCs in vasculature of the yolk sac (ys) in panel D compared with panel C, and by the hemoglobinization of the heart and the common atrial chamber of the heart (h) in panels F and H compared with Mfrn1+/− mice panels E and G.
Figure 3
Figure 3
Deletion of Mfrn1 in adult mice leads to loss of Mfrn1 in hematopoietic tissues. Mfrn1flox/− mice were crossed to mice carrying Mx-Cre. At 3 weeks of age, mice (Mfrn1flox/−;Mx-Cre) were injected with poly(I:C) to induce Cre expression. The mice were analyzed 4-8 weeks later. (A) PCR analysis of Mfrn1 of genomic DNA from thymus (T), spleen (S), liver (L), and bone marrow (Bm) of mice with noted genotypes. (B) RT-PCR analysis of mRNA in the spleens of mice with the noted genotypes using primers specific to exon1 and exon4, which flank exon 2. A wild-type mRNA gave rise to a transcript amplification of 443 bp; a deletion of exon 2 at genomic level gave rise to a transcript amplification of 214 bp. (C) Western analysis of Mfrn1, porin, and tubulin in cytosol, mitochondria, and total bone marrow isolated from mice with the specified genotypes. (D) Bar graph of spleen weight/total weight of mice with the noted genotypes. (E). Hematoxylin and eosin–stained sections of spleen from Mfrn1+/+ mice and Mx-Cre–deleted Mfrn1−/− mice.
Figure 4
Figure 4
Changes in hematologic values after deletion of Mfrn1 in MX-Cre mice. Mice with the noted genotypes were injected with poly(I:C) to induce Cre recombinase. At times subsequent to induction of Cre, blood was taken for analysis of hemoglobin (HGB; A), RBC number (B), hematocrit (C), and MCV (D). Flow cytometric analysis of hematopoiesis in Mfrn1-knockout mice. Groups of Mx-Cre (Control) or floxed Mfrn1/Mx-Cre mice were treated with poly(I:C) and killed 8 weeks later for analysis of hematopoietic tissues. (E) Spleen cell suspensions were treated with ammonium chloride to lyse mature erythrocytes and evaluated for expression of the erythroid marker TER-119, which indicates erythroblast cells. (F-I) Bone marrow cell suspensions were isolated and evaluated for hematopoietic markers as indicated. (F) Selective gating for the hematopoietic stem and progenitor cell compartment (viable cells lacking expression of the lineage markers B220 and CD11b). Mfrn1-deleted mice exhibited increased numbers of erythroid progenitor cells (CD71+TER-119−). (G) Selective gating to exclude B220, CD11b, and TER-119+ cells. It can be seen that Mfrn1-deleted mice exhibit an increase in erythroid progenitor cells (c-kit+CD71+) and proerythroblasts (c-kit−CD71+). We also observed increased numbers of nonviable CD71+ cells (DAPI+CD71+; H) and decreased numbers of B-lineage cells based on B220 expression (I) in Mfrn1-deleted mice. Panels are representative of 6 Mfrn1-deleted mice and 9 control animals.
Figure 5
Figure 5
Deletion of Mfrn1 in hepatocytes leads to increased protoporphyrin IX in mice fed ALA. ALA was added to the drinking water of mice of the noted genotypes. Four weeks later, the ALA-fed mice and control mice were killed and the level of protoporphyrin IX (PPIX) measured in plasma (A), liver (B), and erythrocytes (C). (D) PCR analysis of Mfrn1 deletion in fibroblasts incubated with Tat-Cre. (E) Levels of PPIX in wild-type and Mfrn1-deleted fibroblasts. *P < .05.
Figure 6
Figure 6
Hepatotoxicity in livers of hepatocyte-deleted Mfrn1 animals fed ALA. ALA was added to the drinking water of mice of the noted genotypes (Mfrn1+/+, Mfrn1flox/+, and Alb-Cre;Mfrn1flox/−). Four weeks later, the ALA-fed mice and control mice were killed and liver weight/body weight (A) and spleen weight/body weight (B) were determined. *P < .05.
Figure 7
Figure 7
Histochemical analysis of wild-type and hepatocyte-deleted Mfrn1 livers. Livers from mice fed ALA were harvested at 6 weeks and stained with hematoxylin and eosin (A) or trichrome (B) in Alb-Cre;Mfrn1+/+. (C) Enlarged image from panel B. Alb-Cre;Mfrn1flox/+ were stained with hematoxylin and eosin (D) or trichrome (E). Panel F is an enlarged image from panel E. Alb-Cre;Mfrnlflox/− were stained with hematoxylin and eosin (G) or trichrome (H). Panel I is an enlarged image from panel G. Asterisks in the enlarged images of panels F and I denote areas of fibrosis.
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