The aim of this study was to construct a mammary gland-specific expressional vector pBC1-hLF-Neo for Human Lactoferrin (hLF) gene and then investigate its expression in the mammary gland epithelium cells. The constructed vector contained the 6.2 kb long 5' flank regulation region including promoter, other elements and the 7.1 kb long 3' flank regulation region including transcriptional ending signal of a goat's beta-casein gene. A cassette of Neo gene was also inserted into the vector which gave a total length of 26.736 kb identified by restriction fragment analysis and partial DNA sequencing. The results revealed that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, we transfected the lined vector DNA into the dairy goat's mammary gland epithelium cells and C127 cells of a mouse's mammary epithelium by Lipofectamine. After selection with G418 for 8-10 days, G418-risistant clones were obtained. PCR analysis demonstrated that hLF gene cassette had been integrated into the genomic DNA of G418-risistant clones. After proliferation culture, the two kinds of transgenic cells were cultured in serum-free DMEM-F12 medium with prolactin, insulin and hydrocortisone- a medium capable of inducing recombinant hLF expression. RT-PCR, Western blotting and anti-bacteria bioactivity experiments demonstrated that the constructed mammary gland specific vector pBC1-hLF-Neo possessed the desirable bioactivity to efficiently express and could secrete hLF in both mammary gland cells and have the effect of E. coli proliferation inhibition. Paramount to everything, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.
Animals ; Base Sequence ; Breast Neoplasms ; metabolism ; pathology ; Caseins ; genetics ; Cell Line, Tumor ; DNA, Complementary ; biosynthesis ; genetics ; Epithelial Cells ; metabolism ; Female ; Genetic Vectors ; genetics ; Goats ; Humans ; Lactoferrin ; biosynthesis ; genetics ; Mammary Glands, Animal ; cytology ; metabolism ; Mice ; Molecular Sequence Data ; Mutagenesis, Insertional ; Promoter Regions, Genetic ; genetics