Construction Of Goat Mammary Gland-Specific Expression Vectors

To construct mammary gland-specific expression vectors, 6 β-lactoglobulin (BLG) gene fragments and 1 β-casein gene fragment of expected sizes were amplified from Chinese dairy goat genomic DNA using high fidelity long template PCR system. Partial sequence analyses showed that all 7 gene fragments were more than 99% identical to that of goat and sheep, especially the key regulatory elements in the 5'-end regulatory region of the BLG gene with 100% identity among the three species. This indicates the high fidelity of the DNA polymerase used and the usability of the cloned gene fragments for construction of mammary gland-specific expression vectors. A series of mammary gland-specific expression vectors were constructed by combination of the gene fragments, one of which, known as p205C3, was studied in more detail. The lacZ reporter gene was cloned into the self-constructed vector p205C3 and into two imported vectors pCX and pHC, high expression abilities of which have been demonstrated in both transgenic mice and larger animals. The three recombinant vectors were transfected into goat mammary epithelial cells by using liposome-mediated method, and the transfected cells were stained with a solution containing X-gal following induction with insulin, prolactin and corticosterone. The results showed that the reporter gene was efficiently expressed in the cells transfected with all the three recombinant vectors, but the expression levels in the cells transfected with the self-constructed vector were higher than that with the two imported vectors. The three vectors could not direct the expression of the reporter gene in both COS-1 cells and goat skin fibroblasts. These results suggest that the regulatory sequences of the BLG and p-casein gene cloned from the Chinese dairy goat genomic DNA using high fidelity PCR were usable and the self-constructed goat mammary gland-specific expression vector could be used for generation of mammary gland bioreactors.