Construction of mammary gland-specific expression vectors for human clotting factor IX and its secretory expression...

Two expression vectors comprised of mouse matrix attachment regions (MARs), bovine beta-casein gene sequence, human factor IX (hFIX) minigene, and cDNA, pMCIXm and pMCIX, were constructed for the purpose of a mammary gland bioreactor. A secretory expression system of hFIX protein in milk was made using stearylamine (SA) liposome to transfect plasmid DNA directly into the mammary gland lobule of a lactating goat. The highest production of hFIX in goat milk was 13.7 ng/ml 3 days after transfection, and the hFIX production in the goat mammary gland transfected with pMCIXm containing hFIX minigene was obviously higher than that transfected with pMCIX containing hFIX cDNA. Activity immuno-analysis and the barium citrate absorption method showed that > 90% hFIX protein in milk appeared to be a gamma-glycosylated and biological activity. This result confirmed the validity of the constructed vectors for further transgenic study, and this assay could also find its success in the evaluation of a foreign gene expression and secretion in the milk as a rapid detection system using liposome to transfect DNA directly into the goat mammary gland.