HIGH-LEVEL, STAGE AND MAMMARY-TISSUE-SPECIFIC EXPRESSION OF A CAPRINE KAPPA-CASEIN-ENCODING MINIGENE DRIVEN BY A BETA-CASEIN PROMOTER IN TRANSGENIC MICE

A 5' truncated caprine (ca) kappa-casein-encoding gene (kappa Cas) was fused to the 3' end of a 3' truncated ca beta Cas. The kappa-Cas form comprised the 0.8-kb 3' end of intron 2, the remaining part of the transcription unit containing codons -2 to stop 172, and 0.43 kb of the 3' flanking region. The beta Cas form comprised a 3-kb 5' flanking region and the 5' end of the transcription unit terminating 69 bp dowstream from exon 2 which encodes the 15-amino-acid (aa) signal peptide and the first 2 aa of mature beta Cas. The resulting hybrid gene driven by the peas promoter was expressed in all eight lines of transgenic mice investigated, although at different levels. In two lines, the yield of recombinant (re-) kappa Cas was greater than or equal to 3 mg/ml of milk. The stage- and mammary tissue-specific expression was similar to that of endogenous beta Cas. The re-kappa Cas differed from its goat milk counterpart by the occurrence of four extra aa at the N-terminal end, indicating that the signal peptidase released the beta Cas signal peptide. According to sedimentation analyses of murine milk containing greater than or equal to 3 mg re-kappa Cas/ml, the latter essentially occurred in micelles. Preliminary comparative assays of the behavior of ca alpha(s1)Cas-kappa Cas and alpha(s1)Cas-re-kappa Cas mixtures upon incremental addition of Ca2+ showed that re-kappa Cas had the capacity to protect alpha(s1)Cas against Ca2+-induced precipitation in forming stable micelles. In contrast, the expression of a reconstituted ca kappa Cas gene was in the range of a few mu g re-kappa Cas/ml of milk in the five transgenic lines investigated.