title: Targeted disruption of the mouse ferrochelatase gene producing an exon 10 deletion.
abstract: Protoporphyria is a disease characterized by a deficiency in ferrochelatase, the terminal enzyme in the heme biosynthetic pathway, which catalyzes the chelation of iron and protoporphyrin to form heme. Clinical symptoms arise from an accumulation of protoporphyrin behind the partial enzyme block and include photosensitivity and sometimes hepatobiliary disease. Protoporphyria is described as an dominant disease, yet patients exhibit decreased ferrochelatase activities of 15-30% of normal, not 50% as might be expected. Missense, nonsense, and splicing mutations have been identified in ferrochelatase cDNA from protoporphyric patients. In this study we introduce an exon 10 deletion, an analogous mutation to that described in some protoporphyric patients, into the mouse embryonic stem (ES) cell genome via homologous recombination. Targeted ES cells were confirmed by Southern blot analysis. Expression of wild-type and exon 10-deleted mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA sequencing. Ferrochelatase levels were analyzed by immunoblotting. Ferrochelatase activity was measured by the chelation of zinc and mesoporphyrin, and by the decrease in protoporphyrin accumulation after adding delta-aminolevulinic acid. In the exon 10 +/- ES cells there is expression of both wild-type and exon 10-deleted mRNA, a 50% decrease in cross-reactive material with an anti-ferrochelatase antibody, and an approximate 50% decrease in ferrochelatase activity compared to wild-type ES cells. Therefore, an exon 10 deletion alone is insufficient to decrease ferrochelatase activity to the levels in protoporphyric patients. This suggests that requirement of an additional mutation to decrease the expression of the wild-type allele.
authors.affiliation: University of North Carolina at Chapel Hill 27599-7038, USA.
authors.forename: S T
authors.initials: ST
authors.lastname: Magness
authors.forename: D A
authors.initials: DA
authors.lastname: Brenner
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D002999:Clone Cells; D005091:Exons; D005294:Ferrochelatase / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D018390:Gene Targeting; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011164:Porphyrias / Q000235:genetics; D046351:Protoporphyria, Erythropoietic; D011524:Protoporphyrins / Q000378:metabolism