Expression Of Human Coagulation Factor Ⅸ Gene In Goat Mammary Epithelial Cells And Fibroblast Cells Using Targeting Vetor

Mammary gland bioreactors have been extensively used to prepare active proteins due to such advantages as high output, easy collection of expression products, and exclusion of expression products from circulatory system. Some mammary gland bioreactors have entered commercial production stage, and the current emphasis in this field is dedicated to improving expression efficiency and biological activity of expression products.In this study, the GFP-PGK-Neo was employed as plasmid backbone to construct the homologous targeting vector of human coagulation factor IX. A4798bp fragment upstream of the translation start of goat β-casein was used as a5’homology region, and a3553bp fragment downstream of the open reading frame of goat β-casein as a3’homology region. The human coagulation factor IX cDNA amplified by RT-PCR was located at the down-stream of the5’ homology region. The bGH PolyA was inserted down-stream of the coagulation factor IX gene as a transcription termination signal. The structure of the recombinant plasmid was verified by restriction fragment analysis and DNA sequencing.The targeting vector was introduced into goat mammary epithelial cells and fibroblast cells by electrotrans fection. Stably transfected cells were obtained by G418selection and confirmed by PCR using their genomic DNA. This study lays foundation for preparing transgenic goats by somatic cell nuclear transfer which specifically express human coagulation factor IX in their mammary glands.