PMID: 20646462Jul 22, 2010Paper

Construction of siRNA lentiviral expressing vector targeting pleiotrophin gene and its impact on growth and apoptosis in H446 cells of human small cell lung cancer

Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases
Yong YuHua-cheng Hu

Abstract

To construct a siRNA lentiviral expressing vector targeting PTN (pleiotrophin) gene in human small cell lung cancer H446 cells and to study the RNAi effect on tumor growth and apoptosis. Four pairs of small hairpin RNA specific for PTN were designed, synthesized and cloned into the Plvthm vector. The resulting lentiviral vector containing shPTN was confirmed by DNA sequencing named LV-shPTN. 293T cells were co-transfected with LV-shPTN, pRsv-REV, pMDlg-pRRE and pMD2G, then packed and purified slightly to produce lentivirus. After infecting H446 cells with recombinant lentivirus, PTN expression was determined by real-time RT-PCR and Western blot. Finally the selected lentiviral vector was packed and purified with best interference efficiency in large scale and the titer of virus was determined. The packed virus was used to infect H446 cells, and then the experiment was divided to a normal cell group, a negative control group, a PTN interference group, a chemotherapy group and a combined group with RNAi and chemotherapy. The effect on cell growth and apoptosis of H446 cells infected with high titer virus was analyzed using MTT and FCM. DNA sequencing analysis confirmed that shPTN lentiviral vector was successfully established as ...Continue Reading

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