High-level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence

In our previous study, transgenic mice were generated that expressed human lactoferrin (hLF) in milk using cDNA under control of the 2 kb bovine beta- casein promoter. The expression level of the protein in milk of 7 mice rang ed from 1 to 200 mu g/ml; 1 to 34 mu g/ml in 6 mice and 200 mu g/ml in 1 mo use. With the aim of inducing higher expression of the protein, we construc ted an expression cassette comprised of 10 kb of the bovine beta-casein gen e promoter and the hLF genomic sequence in place of the cDNA. The hLF genom ic sequence of about 27 kb, spanning 23 kb of the entire coding region and 4 kb of the 3'-flanking sequence, was placed downstream the bovine beta-cas ein promoter, In total, 8 transgenic mice were generated from 31 mice (tran sgenic rate of 25.8%) born from the embryos microinjected with the 40-kb hL F expression cassette. Mammary-specific expression of the transgene was add ressed by performing Northern hybridization of the total RNAs hom various t issues of transgenic mice. Immunoblot analysis showed that the recombinant protein expressed in milk has the same molecular weight as the native prote in. The amount of the the protein in milk of 5 mice ranged from 60 to 6,600 mu g/ml when judged by ELISA analysis. Three mice expressed the protein at the level higher than 500 mu g/ml. These data suggest that the genomic lac toferrin sequence represents a valuable element for the efficient expressio n of the protein in milk of transgenic animals.