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Figures
- Fig. 1.
Phenotype of the RBC16 mouse mutant. (A) Peripheral blood smears of 7-week-old wild-type (WT) and RBC16 heterozygous (+/M) mice show microcytic hypochromic red cells and prominent target cells. (B) H&E staining of sectioned spleens harvested from 4-month-old wild-type and heterozygous mice at both low (×40) and high (×200) magnification. (C) FACS plot of spleen cells stained with Ter-119 and CD71. Gated Ter-119+ CD71hi cells reveal a significant increase in early erythroblast (E.i) populations in the mutant. (D) Red cell half-life measured in vivo using biotinylation; n=4. Whole blood haem (E) and serum ferritin (F) quantification. Values are mean±s.d; n=4. *P<0.05, **P<0.01.
- Fig. 2.
Characterisation of the W373X mutation. (A) Fine SNP mapping of 40 mice at 15 SNP markers localised the RBC16 mutation between 56.46 Mb and 60.98 Mb on chromosome 16. Light grey indicates homozygous for Balb/c; dark grey indicates heterozygous for Balb/c and C57BL/6. (B) Sanger sequencing of heterozygous RBC16 gDNA shows the G→A substitution in Cpox (arrowhead), whereas in heterozygous cDNA, only the wild-type sequence is visible. (C) Quantitative RT-PCR for Cpox mRNA expression in the liver. Values are mean±s.d., n=4; **P<0.005. (D) Western blot of the CPOX protein from liver lysates of WT and +/W373X mice. (E) Wild-type (WT) and homozygous (W373X/W373X) embryo littermates dissected at day 9.5, showing yolk sac (top) and whole embryo formation (bottom).
- Fig. 3.
Porphyrin studies in Cpox+/W373X mice. (A) Baseline urinary and (B) faecal porphyrins of 5-month-old RBC16 wild-type (WT) and heterozygous (+/W373X) mice of each gender. ‘Other’ represents porphyrins too low in concentration to accurately distinguish. (C) Isomer ratio of faecal coproporphyrin III to I (CIII:CI) and (D) baseline urinary PBG levels measured in female wild-type (WT) and heterozygous (+/W373X) mice. Values are mean±s.d.; n=3. **P<0.01, ***P<0.001, n.s., not significant.
- Fig. 4.
Effects of fasting on female Cpox+/W373X mice. (A) Urinary porphyrins of 4-month-old RBC16 wild-type (WT) and heterozygous (+/W373X) female mice measured before and after (+fast) fasting period. (B) Isomer ratio of faecal coproporphyrin III to I (CIII:CI) measured before and after (+fast) fasting period. (C) Urinary PBG quantification before and after (+fast) fasting period. (D) Quantitative RT-PCR for Alas-1 and (E) Cpox mRNA expression in the liver before and after (+fast) fasting period. Values are mean±s.d.; n=3 for A-C and n=4 for D,E. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.
- Fig. 5.
Effects of phenobarbital treatment on female Cpox+/W373X mice. (A) Faecal porphyrins of 4-month-old wild-type (WT) and heterozygous (+/W373X) female mice measured before and after (+Pb) phenobarbital treatment. ‘Other’ represents porphyrins too low in concentration to accurately distinguish. (B) Isomer ratio of faecal coproporphyrin III to I (CIII:CI) measured before and after (+Pb) phenobarbital. (C) Urinary porphyrins of wild-type (WT) and heterozygous (+/W373X) female mice measured before and after (+Pb) phenobarbital treatment. (D) Urinary PBG quantification of wild-type (WT) and heterozygous (+/W373X) female mice measured before and after (+Pb) phenobarbital treatment. (E) Quantitative RT-PCR for Alas1 and (F) Cpox mRNA expression in the liver before and after (+Pb) phenobarbital treatment. Values are mean±s.d.; n=3 for A-C and n=4 for D,E. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.
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