Isolation and functional characterization of buffalo (Bubalus bubalis) -casein promoter for driving mammary epithelial cell-specific gene expression

Abstract

Therapeutic proteins are produced in microbes, mammalian cell lines, and body fluids by applying recombinant DNA technology. They are required for compensating the deficiency of essential proteins in patients. Animal bioreactors producing such valuable bio-pharmaceuticals in body fluids have lately emerged as efficient and cost-effective expression systems. Promoters, along with other regulatory elements of genes coding for milk proteins, have been cloned from few species for directing the expression of desired proteins in the milk of farm animals. However, buffaloes, which are the second largest source of milk production in the world, have remained unexplored for such use. Since mammary epithelial cell-specific β-casein is the most abundantly expressed protein found in buffalo milk, we have isolated the promoter region and the transcriptional regulatory element along with exon 1, Intron 1 and partial exon 2 of the β-casein gene from the genome of the Indian river buffalo (Bubalus bubalis) and have characterized the same (GenBank accession no. KF612339). Mammary epithelial cells of buffalo and human (MCF7) expressed Enhanced green fluorescent protein (EGFP) upon transfection with the construct where egfp was cloned under the β-casein promoter. Transfected HEK-293 cells failed to express EGFP. Transgenic female mice generated using this construct expressed EGFP in the milk gland during lactation, without leaky expression in any other organs. This promoter also drove expression of recombinant human Interferonγ suggesting its use for expressing recombinant bio-pharmaceuticals in the milk of buffalo or other farm animals. Additionally, this may also allow breast gland-specific gene expression for remediation of breast gland-associated diseases.