Establishment Of Mammary Epithelial Cell Lines And RNA Interference-mediated Knockdown Of DGAT1Decreases TG Content Of Cow

Chinese Holstein dairy cow is the main dairy cow in China. With the development of Chinesemilk industry, more attention is being placed on the factors that might affect milk quality,especially milk fat and milk protein content. The study established a culture system for two typesof bovine mammary epithelial cell line, high fat high protein mammary epithelial cell line(MEC-HH) and low fat low protein mammary epithelial cell line (MEC-LL) in vitro, and elucidatethe unique characteristics of these cell lines. Liposome mediated-RNA interference technologywas employed to knockdown endogenous gene DGAT1expression in bovine mammary epithelialcells and biological function changes of cells were analyzed.In the bovine species, milk production is well known to correlate with mammary tissue.However, most advanced techniques in optimizing milk production relied on improvements ofbreeding and husbandry practices. Our study established two mammary epithelial cell lines fromChinese Holstein cows whose milk contained high fat high protein and low fat low protein withtissue culture method and the MECs were thoroughly characterized via morphology, growthcurves, staining analysis of cells nucleus, RT-PCR and immunocytochemistry. The comparisonabout the two cell lines were done by detection of TG contents and total protein contents intracellular, and the isolated MECs have been used for investigation of gene function about milk fatand protein and lipometabolism mechanism study.DGAT1(diacylglyceroltransferase-1) expresses in nearly all tissues, including the mammarygland tissue. Mice lacking DGAT1decreased triglyceride content in mammary tissue, and areresistant to diet-induced obesity and diabetes mellitus. However, the function of DGAT1has notbeen verification by RNAi level yet. The authors examined the function of DGAT1by RNAi inthis study. Liposome mediated-RNA interference technology was employed to knockdownendogenous gene DGAT1expression in bovine mammary epithelial cells and biological functionchanges of cells were analyzed. The mRNA and protein levels, intra-cellular triglyceride（TG）content and total protein were detected by real-time PCR, western blot, TG-kit and ultravioletspectrophotometer after RNAi treatment.The results indicated that knockdown of DGAT1 expression significantly inhibited TG content in bovine mammary epithelial cells. The study maybe further defined that DGAT1is essential for triglyceride synthesis in bovine mammary tissue.