Production of transgenic dairy goat expressing human alpha-lactalbumin by somatic cell nuclear transfer

摘要

Production of human alpha-lactalbumin (h alpha-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat beta-lactoglobulin (beta LG) promoter, the h alpha-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of h alpha-LA was investigated. Both the h alpha-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency a parts per thousand 60-70 %) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these h alpha-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0 %, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried h alpha-LA and the h alpha-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.