Generation of the Hepc1 knockout mice and phenotypic exploration. (A) Schematic representation of the targeting strategy. The structure of the Usf2/Hepc1/Hepc2 locus is shown at the top,6 the targeting construct in the middle and the resulting targeted allele at the bottom. The genes are represented by colored rectangles and the arrows represent the direction of the transcription. Restriction sites for HindIII (H) and BglII (B), used for Southern blot genotyping, and probe location are indicated. The arrowheads indicate the location of the primers designed for the RT-PCR (B). The scheme is not drawn to scale. HF indicates homologous fragments. (B) Level of specific Hepc1, Hepc2, and Usf2 transcripts in the livers of controls, Usf2–/– and Hepc1–/– mice age 6 to 8 months, as measured by RT-PCR. Following PCR, the amplified products (171 bp for Hepc1 and Hepc2 and 250 bp for β-actin and Usf2) were separated by electrophoresis on 1.5% agarose gel. M indicates 100-bp DNA ladders. The vertical dotted lines indicate assembly of noncontiguous lanes. (C) Perls staining of liver, pancreas, heart, and spleen sections. Typical liver, pancreas, and spleen sections (original magnification ×10) from Hepc1+/+ and Hepc1–/– mice age 4 months. Non-heme iron stains blue. (D) Perls staining of liver sections (original magnification ×10). Left, typical liver section of a 2-month-old Hepc1–/– animal. CL, centrolobular; PP, periportal. Right, liver sections from 2-month-old Hepc1–/– and Hepc1–/– animals submitted to an iron-rich diet for 14 days.
