shRNA稳定干扰Raptor基因表达的小鼠胸腺上皮细胞系的建立

目的: 建立Raptor基因稳定干扰的小鼠胸腺上皮细胞系(mouse thymic epithelial cell line 1, mTEC1)。方法: 根据Sigma公司数据库Raptor基因的短发夹RNA(short hairpin RNA,shRNA)序列,在其3′和5′端添加pLKO.1 puro慢病毒载体酶切位点序列,合成shRNA序列,插入载体。将pLKO.1 puro shRNA Raptor慢病毒载体与包装质粒混合,共同转染293T细胞,收集病毒颗粒感染mTEC1,再用嘌呤霉素对其进行筛选,最后用免疫印迹法检测Raptor,mTOR,磷酸mTOR(p mTOR)以及mTOR下游分子核糖体S6激酶(ribosomal S6 kinase, p70S6k),磷酸化p70S6k(p p70S6k)的表达水平。同时采用细胞计数和TUNEL法分别检测细胞增殖与凋亡。结果： 合成的Raptor基因shRNA序列成功插入至经Age Ⅰ和EcoR Ⅰ酶切的pLKO.1 puro载体,经测序表明序列正确;免疫印迹结果显示,pLKO.1 puro shRNA Raptor感染mTEC1后,Raptor、mTOR及其下游p p70S6k蛋白表达明显降低,但细胞增殖及凋亡无明显变化。 结论： 成功构建Raptor shRNA慢病毒表达载体,建立稳定的Raptor基因敲降的小鼠mTEC1。

Establishment of mouse thymic epithelial cell line stably knocking down the expression of Raptor gene by shRNA

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.1 puro lentiviral vector cleavage site sequence at its 3′ and 5′ ends and inserted into pLKO.1 puro lentiviral vector.The pLKO.1 puro shRNA Raptor lentiviral vector was co transducted 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(p mTOR), and the mTOR downstream molecule ribosomal S6 Kinase (p70S6k) and phosphorylated p70S6k(p p70S6k) 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.1 puro vector which digested by Age Ⅰ and EcoR Ⅰ and the pLKO.1 puro shRNA Raptor was successfully constructed. The protein expression levels of Raptor, p mTOR and p p70S6k 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.