Iron status in mice carrying a targeted disruption of lactoferrin

Abstract

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins present in milk, mucosal secretions, and the secondary granules of neutrophils. While several physiological functions have been proposed for lactoferrin, including the regulation of intestinal iron uptake, the exact function of this protein in vivo remains to be established. To directly assess the physiological functions of lactoferrin, we have generated lactoferrin knockout (LFKO(-/-)) mice by homologous gene targeting. LFKO(-/-) mice are viable and fertile, develop normally, and display no overt abnormalities. A comparison of the iron status of suckling offspring from LFKO(-/-) intercrosses and from wild-type (WT) intercrosses showed that lactoferrin is not essential for iron delivery during the postnatal period. Further, analysis of adult mice on a basal or a high-iron diet revealed no differences in transferrin saturation or tissue iron stores between WT and LFKO(-/-) mice on either diet, although the serum iron levels were slightly elevated in LFKO-/- mice on the basal diet. Consistent with the relatively normal iron status, in situ hybridization analysis demonstrated that lactoferrin is not expressed in the postnatal or adult intestine. Collectively, these results support the conclusion that lactoferrin does not play a major role in the regulation of iron homeostasis.

FIG. 1.

Generation of LFKO^−/− mice. (A) Targeting strategy. A schematic diagram of the targeting construct is shown in the top panel. Numbered boxes represent exons. The wild-type allele and the targeted allele are shown in the middle and bottom panels, respectively. After homologous recombination, the targeted allele contains an additional EcoRI site introduced with the neomycin resistance gene cassette. A 5′ probe located outside the region of homology detects a 10.6-kb fragment in the wild-type allele and a 6.6-kb fragment in the targeted allele. HSV-TK, herpes simplex virus thymidine kinase gene. (B) Genotype identification by Southern blot analysis. Genomic tail DNA from offspring from heterozygote intercrosses was digested with EcoRI and probed with a 5′ probe located outside the region of homology. In WT mice, a single radioactive band at 10.6 kb, corresponding to two normal alleles, is detected (third lane). In LFKO^+/− mice, two hybridizing bands, representing the 10.6-kb normal allele and the 6.6-kb targeted allele, are detected (second lane). LFKO^−/− mice are detected by the presence of a single radioactive band at 6.6 kb, corresponding to two targeted alleles (first and fourth lanes). (C) Genotype identification by PCR analysis. A PCR product of 515 bp represents the WT allele, whereas a product of 575 bp represents the targeted allele. WT mice (first and second lanes), LFKO^+/− mice (third lane), and LFKO^−/− mice (fourth lane) are identified. (D) Western immunoblot analysis of mouse milk samples. Western immunoblot analysis was performed by using specific antisera directed against murine lactoferrin. A single immunoreactive band migrating at the size expected for lactoferrin is detected in the milk samples from WT mice (first lane) and LFKO^+/− mice (second lane) but not in the milk samples from LFKO^−/− mice (third lane).

FIG. 2.

Intestinal cell morphogenesis in LFKO^−/− mice. Intestinal sections from WT mice (left panels) and LFKO^−/− mice (right panels) were analyzed at postnatal day 18. Sections of the proximal duodenum were stained as follows. (A and B) Anti-human IFABP; specificity, enterocytes. Staining (brown) is confined to the villus enterocytes. Arrows indicate negatively stained goblet cells. (C and D) Antiserotonin; specificity, enteroendocrine cells. Arrowheads indicate representative stained cells (brown). (E and F) Anti-human lysozyme; specificity, Paneth cells. Staining (brown) is confined to the crypts. (G and H) Alcian blue; specificity, goblet cells. Positive cells (blue) are detected in the villi. No staining was observed in control sections incubated with nonimmune serum in place of primary antibody (data not shown). Stained sections are representative of at least seven mice analyzed for each genotype group. Magnifications: A to D, ×200; E to H, ×320. Scale bars, 5 μm.

FIG. 3.

In situ hybridization analysis of lactoferrin expression in mouse tissues. Sections were incubated with a specific ³⁵S-labeled antisense (A, C, and E) or sense (B, D, and F) murine lactoferrin probe. Dark-field illumination shows postnatal day 18 duodenum (A and B), adult duodenum (C and D), and estrogen-primed uterus (E and F). Magnification, ×100. Scale bars, 20 μm.