Cloning and Molecular Characterization of Porcine β-casein Gene (CNS2)

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, β-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine β-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine β-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP β. In addition, the first intron of the porcine β-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP β, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine β-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine β-casein gene. The β-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine β-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine β-casein gene activity.