Construction Of Transgenic Mammary Epithelial Cells For Sfat-1

Mammary gland epithelial cells have special functions of synthesis and lactation .These functionsmake them mammary bioreactor harrow cell. Experimental model made from mammary gland epithelialcells could actually response many biology mechanisms ,such as mammoplasia ,development ,the startand maintenance of lactation .The model could also validate if the construction of tissue specificityexpression vector is rational and effective .Mammary gland epithelial cells and the experimental modelsrelate to molecular biology, endocrinology developmental biology , cell biology , biochemistry and andother fields .It is significant to the gland bioreactor ,and the research of the lactoprotein geneexpression .The ratio ofω-6/ω-3 fatty acid has effects on health and chronic disease.ω-6 PUFAs can beobtained through the food directly ,or synthesized in the human body .Mammals are lack of the enzymethat could synthesizeω-3 PUFAs, soω-3 PUFAs can be obtained through the food only. In moderndiet ,the ratio ofω-6/ω-3 is very high, it have impact on the health of modern people. In this case, it isnecessary to enrich the food supply withω-3 PUFAs to reduce the ratio and improve the nutritionalcomposition of diet. The fat-1 gene encodesω-3 fatty acid desaturase that could convert theω-6 fattyacid to theω-3 fatty acid.The experimental material of this study was health Holstein cow mammary gland tissue fromslaughterhouse.We used different methods to culture the primary mammary epithelial cells,successfully cultured bovine mammary epithelial cells(BMEC).The established mammary epithelial cellswere identified by immunofluorescence staining. The results showed that the large numbers of mammaryepithelial cells were successfully separated from mammary tissue inoculation. The cells were incubatedwith DMEM-F12 and 20% fetal bovine serum,insulin(1μg/mL),transferring(5μg/mL),pregnendione(1μg/mL),hydrocortisone(1μg/mL).Mammaryepithelial cells and fibroblasts were mixed grown. The boundaries between the two different cells wereclear during primary culture. But the boundaries were not so clear after passage. Mammary epithelialcells have different morphology, most of them were cobblestone and polygon..Mammary epithelial cells were transferred using vectors constructed in our lab: two mammarygland-specific vectors pCB1F1E (8976bp) pCB2F1E(9101bp),and two negative control vectors pCB1E(7823bp) and pCB2E (7948bp).The positive transgenic cells were obtained The PCR test proved thatfat-1 gene were successfully integrated within the genome.