Study On Construction Of Mammary Gland Specific Expression Vector For Ssβ-gly And Its Transfection Of Bovine Fetal Fibroblasts Mediated By Liposome

Producing bioactive proteins through mammary gland bioreactor is a hot focus in the field of biology, and the key question is the construction of expression vector in mammary gland. Beta-lactoglobulin (BLG) is the main protein in ruminant milk. For the purpose of constructing mammary gland specific expressional vector, we use the updream regulation sequence of bovine BLG to regulate lacS gene to express in mammary gland. Positive clone cells are selected and purified by the expression Neo gene and report gene EGFP. So the cells can be used as sources to create transgenic animals by nuclear transfer techniques. In our studies, main involved the construct of mammary gland-expression vector, establishment and sex identification of bFF(bovine fetal fibroblasts), optimization of transfection efficiency of bFF mediated by liposome. The results of research are as following:Firstly, a 0.92kb bovine beta-lactoglobulin gene 5′upstream regulatory sequence and 1.49kb lacS gene coding sequence was amplified by PCR technology. Bovineβ-lactoglobulin gene 0.92kb promoter, 1.49kb lacS gene, pIRES2-EGFP and pUC19 vector were used to construct a mammary gland-specific expression vector—pBLI, which contained the Neo gene and the EGFP gene as markers regulated by CMV promoter for expression in a non-tissue specific mode and the lacS gene regulated by bovineβ-lactoglobulin promoter for expression specifically in mammary gland.Secondly, Fibroblasts derived from 2～3 month old bovine fetus were successfully cultured with a normal tissue culture method. The cultured cells were analyzed by their morphology, growth curve and S4 gene sex identification. Results demonstrated that these cultured cells were typical fibroblasts with normal morphology,and donor bovine of cell is female. The sensitivity studies of bFF against G418 concentration in DMEM/F12 media indicated as high as 800μg/ml of G418, and the concentration of G418 can be reduced to 300μg/ml during the cell maintenance stage.Finally, the linear pBLI plasmid vector coated with liposomere agent,was transferred into the bFF to validate pBLI. Such parameter effecting transfection efficiency as cell confluency, DNA amount, the morphology of vector and serum were analyzed, and transfection efficiency was optimized by fluorescent microscope. It was found that the highest efficiency was achieved with 1.0μg linear DNA plasmid, the ratio of liposome and...