Cloning of 5′ regulatory element of bovine β-lactoglobulin gene and its utilization in generation of mammary bioreactors

摘要

To obtain a regulatory element for generating mammary bioreactors, a DNA fragment derived from bovine β-lactoglobulin (BLG) gene was cloned, which consisted of a 650-bp 5′ flanking sequence, exon Ⅰ, intron Ⅰ and exon Ⅱ. A 661-bp region of the cloned fragment, consisting of the 650-bp 5′ flanking sequence and a non-coding sequence of 11 bp downstream of the transcription initiation site, was used as a regulatory element to combine with human growth hormone (hGH) gene to generate a bovine BLG/hGH fusion construct, which was then introduced into cultured primary mammary epithelial cells of goat for transient expression of hGH gene. It was demonstrated that the hGH gene was able to express following hormone induction and the expressed product was able to be secreted into the medium. The bovine BLG/hGH fusion construct was also used to generate transgenic mice by microinjection. Subsequently, five transgenic mice were generated. The hGH in milk by one transgenic female mouse was 420 蘥/mL, while the content of hGH in serum was 0.051 μg/mL only. This indicated that the cloned regulatory element in this experiment could make the expression of target gene occur almost specifically in mammary gland and the expressed product could be secreted with milk.