Genetic modification of bovine beta-casein and its expression in the milk of transgenic mice

Genomic vectors containing mutant bovine beta-casein with putative glycosylation sites were constructed to study the functional properties of glycosylated beta-casein and its possible effects in milk. The mutation was performed by PCR-based site-directed mutagenesis. The tripeptide sequence, Asn-X-Ser, was generated between Asn(68) and Asn(73) in mature beta-casein. The resulting beta-casein mutants were designated pCJB68 and pCJB6873. pCJB68 carries a substitution of Ser io for Leu(70) (Asn(68)-Ser(69)-Ser(70)-Pro(71)), and pCJB6873 carries a substitution of Ser(70)-ser(71) for Leu(70)-Pro(71) (Asn(68)-Ser(69)-Ser(70)-Ser(71)). The two mutated genomic constructs were placed under control of the bovine alpha-lactalbumin promoter, and lines of mice expressing the pCJB68 and pCJB6873 have been established. The milk from transgenic mice contained bovine beta-casein at levels up to 2-3 mg/mL. N-Linked glycosylation of bovine beta-casein in the pCJB6873 line was confirmed by peptide-N-glycosidase F treatment, but glycosylation of bovine beta-casein did not occur in pCJB68 mice. In addition, mouse casein micelles containing glycosylated bovine beta-casein showed the largest median diameter and rough outer surface, compared to normal mouse casein micelles and micelles from transgenic milk containing bovine beta-casein.