kappa-casein-deficient mice fail to lactate

Abstract

Acquisition of milk production capabilities by an ancestor of mammals is at the root of mammalian evolution. Milk casein micelles are a primary source of amino acids and calcium phosphate to neonates. To understand the role of kappa-casein in lactation, we have created and characterized a null mouse strain (Csnk-/-) lacking this gene. The mutant kappa-casein allele did not affect the expression of other milk proteins in Csnk-/- females. However, these females did not suckle their pups and failed to lactate because of destabilization of the micelles in the lumina of the mammary gland. Thus, kappa-casein is essential for lactation and, consequently, for the successful completion of the process of reproduction in mammals. In view of the extreme structural conservation of the casein locus, as well as the phenotype of Csnk-/- females, we propose that the organization of a functional kappa-casein gene would have been one of the critical events in the evolution of mammals. Further, kappa-casein variants are known to affect the industrial properties of milk in dairy animals. Given the expenses and the time scale of such experiments in livestock species, it is desirable to model the intended genetic modifications in mice first. The mouse strain that we have created would be a useful model to study the effect of kappa-casein variants on the properties of milk and/or milk products.

Fig. 1.

Targeting of the κ-casein gene. (A) Upon targeting, exon 1 and 3 kb of upstream region are replaced with a cassette containing LoxP, 3′ Hprt gene sequences, and the neomycin gene. (B) Southern blot analysis of targeted ES cells showing a 7.4-kb BamHI fragment hybridizing to a probe external to the targeting vector. (C) Northern blot analysis of κ-casein gene expression in the mammary gland (postpartum day 0) showing lack of transcript in Csnk^−/− mice and gene dosage effect in Csnk^+/− mice in comparison with the wild-type mouse. β-Casein gene expression in the absence of κ-casein gene expression is not affected. Blots were reprobed with β-actin cDNA for normalization of total RNA loaded.

Fig. 2.

Calcium-sensitive caseins aggregate in the Csnk^−/− mammary gland (postpartum day 0). (A) Toludine blue staining of semithick plastic sections of wild-type mammary gland shows well formed alveoli with clear lumina (arrow). (B) In Csnk^−/− mice, alveolar lumina (arrow) are blocked with proteinaceous precipitates. (D and G) Sections of Csnk^−/− and wild-type mammary gland, respectively, stained with Alexa Fluor 350-labeled antibodies against mouse β-casein protein. (C and F) Staining of nuclei with propidium iodide (PI). E and H are overlaps of Alexa Fluor 350 and PI staining. These observations confirm that aggregates in Csnk^−/− lumina (arrow) are other caseins. (Scale bars, 10 μm.)

Fig. 3.

Toludine blue staining of mammary gland sections of wild-type and Csnk^−/− mice at midpregnancy (16.5 dpc). (A) Alveolar lumina (arrow) of the wild type lack precipitates. (B) Precipitates have started appearing in Csnk^−/− alveolar lumina. (Scale bars, 5 μm.)

Fig. 4.

Calcium-sensitive caseins are organized into micelle-like structures in the absence of κ-casein. (A) Immunogold staining of the wild-type mammary gland (postpartum day 0). (B) Immunogold staining of a Csnk^−/− gland (postpartum day 0). Multiple micellar like structures are present in the secretory vesicles of Csnk^−/− glands as in that of the wild type. However, they are seen coalescing after secretion in Csnk^−/− mice, unlike in the wild type. Anti-mouse casein serum raised in rabbits was used for immunogold staining. L, lumen; M, micelles; SV, secretory vesicles; AM, apical membrane. (Scale bars, 0.1 μm.)

Fig. 5.

Casein micelles show coalescence in Csnk^+/− milk. (A) AFM examination of Csnk^+/+ micelles; scan size is 2.5 μm. (B) Some of the Csnk^+/− micelles coalesce to form larger, irregularly shaped structures.