Construction and identification of lentiviral vector of the RNA interference of human Nanog gene

Abstract

Objective To construct a lentiviral vector of RNAi of Nanog,which lay a foundation to the further explore its role in biological behaviour of gliomas by lentivirus-mediated silencing Nanog.Methods The effective sequence of shRNA targeting Nanog gene was designed according to the Elbashir' s criteria and the RNAi vector rule.The complementary DNA containing both sense and antisense oligo DNA of targeting sequence of Nanog was synthesized and cloned into pLKO.1-puro vector,to construct the lentiviral vector which expressed shRNA,and it was confirmed by PCR identification and DNA sequencing.The pLKO.1-Nanog-shRNA,pCMV-dR 8.2 dvpr and pCMV-VSV-G were cotransfected into 293T incasing cells.Harvesting recombinant lentivirus and detecting viral titer that was able to express shRNA targeting Nanog gene.Results The recombinant lentiviral vector expressing shRNA targeting Nanog gene was successfully constructed and identified by polymerase chain reaction (PCR) and DNA sequencing.The recombinant lentivirus were harvested from supernatant of 293T cells with a viral titer of 106 TU/ml.Conclusion The lentivirus RNAi vector of Nanog was constructed successfully,which provide a basis for investigation of the role of Nanog and the mechanism of Nanog transcriptional regulation in gliomas in vitro and in vivo. Key words: Glioma; Nanog; Lentiviral vector; RNA interference