广东医学2012，Vol.33Issue(7)：903-907,5.
人源性Jagged1RNAi慢病毒载体的构建及鉴定
Construction and identification of the human Jagged1-targeted RNAi system on lentiviral vector.
摘要
Abstract
Objective To construct human Jaggedl - RNAi lentiviral vector, and to observe the knocking - down effect in tongue cancer cells. Methods Human Jaggedl information was retrieved in GenBank and for designation of Jaggedl silence targets. After BLAST homology comparison test, synthetic double - stranded DNAoligo was constructed. Plas-mid RNA interference pAJ - U6 - shRNA - CMV - eGFP/PuroR was subsequently linearized, digested with restriction enzymes and recombined with the synthetic double - stranded DNAoligo. The recombinant plasmid was transfected into E. Coli competent cells and selected for positive clones. 293T cells was infected with the Jaggedl RNAi expression plasmid pAJ - U6 - Jaggedl - shRNA - CMV - eGFP/PuroR. Three lentiviral systems were co - transfected into 293T cells. Lentiviral vector packaged from 293T cells were purified. Tca8113 and Cal27 cells were transfected with the to assess the transfection efficacy. Real time PCR and Western Blot were used to detect intracellular Jaggedl gene and protein expression. Results The Jaggedl - RNAi lentiviral vector was successfully constructed. Intensive green fluorescent cells can be seen in Tca8113 and Cal27 cells, suggesting effective infection. Intracellular Jaggedl gene and protein expression were significantly reduced according to Real time PCR and Western Blot in transfected cells. Conclusion The Jaggedl - RNAi lentiviral vector is successfully constructed, providing foundation for further research of the mechanism of Jaggedl and Notch signaling pathway in the development of human tongue cancer squamous cell carcinoma.关键词
Notch信号/Jagged1/舌癌/RNA干扰/慢病毒Key words
Notch signaling/Jaggedl/tongue cancer/RNA interference/lentiviral引用本文复制引用
张同韩,刘海潮,梁玉洁,梁立中,廖贵清,吴纪楠,黄洪章..人源性Jagged1RNAi慢病毒载体的构建及鉴定[J].广东医学,2012,33(7):903-907,5.基金项目
国家自然科学基金资助项目(编号:30772442) （编号:30772442）
