The Transfection Of Human Lactoferrin Gene Into Goat Mammary Epithelial Cell Lines And Establishment Of The Transfected Cell Lines

Nowadays, the study of mammary gland bioreactor is one of the most popular problems in life and biotech areas. In the theory, it is possible to transfer fusion gene vector specially expressed in mammary gland into mammary epithelial cells, but the successful report is scarce.On the basis of the successful technique of somatic nuclear transfer and mammary gland bioreactor, this thesis research on constructing mammary gland-specific expressional vector systematically, establishing goat mammary epithelial cell lines in vitro, transferring foreign genes into cultured cells and establishing transfected cell clones, of which are four main steps to establish the transgenic cloning research methods and technology.1 Construct mammary gland-specific expressional vectorFor the purpose of constructing mammary gland-specific expressional vector BLC-14, we used cDNA sequence of human lactoferrin gene, 5' and 3' flank regulatory sequence of β-lactoglobulin gene, neo^r gene and the sequence of enhancer which had harboured in our laboratory.2 Establish goat mammary epithelial cell lines in vitroPrimary goat mammary cells could be achieved by tripsin digested lacteal and collegenase I dissociated goat mammary gland tissue form the lactating goat, then the growth of cells in medium DMEM/F₁₂ containing 10% fetal calf serum. Comparison of the growth of cells obtained by two different methods, we acknowledged that the cells obtained through tripsin digesting began to be decrepitute after it kept normal characterstics by 10th passages. However the cells obtained through collegnase I dissociating could keep proliferate vigorously even in the vitro 25th culture except for some fibroblast and epithelial cells complex culture existing. By the methods of tripsin digesting and attaching repeatly, the pured mammary epithelial cell line could be...