Establishment of mouse thymic epithelial cell line stably
knocking down the expression of Raptor gene by shRNA
MIN Yu-jiao1,2, WU Hao-jie1,2, WANG Hui1,2, PAN Zi-hui1,2, SHAO Qi-xiang1,2
（1. Department of Immunology, School of Medicine, Jiangsu University, Zhenjiang Jiangsu 212013; 2. Reproductive Sciences Institute, Jiangsu University, Zhenjiang Jiangsu 212001, China)
（1. Department of Immunology, School of Medicine, Jiangsu University, Zhenjiang Jiangsu 212013; 2. Reproductive Sciences Institute, Jiangsu University, Zhenjiang Jiangsu 212001, China)
Abstract：Objective: To establish a mouse thymic epithelial cell line(mTEC1), which Raptor gene was stably knocked down. Methods: The short hairpin RNA(shRNA) sequence of Raptor gene was synthesized according to its sequence in Sigma database and added the pLKO.1puro lentiviral vector cleavage site sequence at its 3′ and 5′ ends and inserted into pLKO.1puro lentiviral vector.The pLKO.1puroshRNARaptor lentiviral vector was cotransducted with packaging plasmid into 293T cells and the virus particles were harvested. The mTEC1 was transfected with virus and selected by puromycin. The proteins expression levels of Raptor, mTOR, phosphorylated mTOR(pmTOR), and the mTOR downstream molecule ribosomal S6 Kinase (p70S6k) and phosphorylated p70S6k(pp70S6k) were determined by Western blottting. Cell proliferation and apoptosis were detected by cell counting and TUNEL assay. Results: The correct shRNA sequence of Raptor gene was inserted into pLKO.1puro vector which digested by Age Ⅰ and EcoR Ⅰ and the pLKO.1puroshRNARaptor was successfully constructed. The protein expression levels of Raptor, pmTOR and pp70S6k was decreased significantly. The cell proliferation and apoptosis showed no significant differences. Conclusion: The Raptor shRNA lentiviral expression vector was successfully constructed and the stable Raptor gene knockdown mTEC1 was established.
闵玉娇1,2, 吴皓杰1,2, 王荟1,2, 潘子辉1,2, 邵启祥1,2. shRNA稳定干扰Raptor基因表达的小鼠胸腺上皮细胞系的建立[J]. 江苏大学学报:医学版, 2019, 29(02): 108-112.
MIN Yu-jiao1,2, WU Hao-jie1,2, WANG Hui1,2, PAN Zi-hui1,2, SHAO Qi-xiang1,2. Establishment of mouse thymic epithelial cell line stably
knocking down the expression of Raptor gene by shRNA. Journal of Jiangsu University(Medicine Edition), 2019, 29(02): 108-112.
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2019, Vol. 29 
