Package and identification of a lentiviral vector of RNA interference containing EZH2 gene

Objective Peripheral T-cell lymphomas (PTCLs) originated from mature,post thymic T cells are a rare heterogeneous group of clinically aggressive non-Hodgkin lymphoma (NHL) with a dismal prognosis.This study was aimed to package and identify a lentiviral vector of RNA interference (RNAi) targeting enhancer of zeste homolog 2 (EZH2),would lay the foundation for the future study.Methods After transformation into competent E.coli bacteria,the candidate clones were identified by DNA sequencing.The EZH2-short hairpin RNA (shRNA) plasmid and the two packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral vector.The Hut78 cells were infected with the lentiviral vector obtained and the transfection efficiency was assessed under the fluorescent microscope.Then the expression of EZH2 in the transfected cells was determined by RT-qPCR and Western Blot.Results The lentiviral RNAi vector for the EZH2 gene was packaged successfully.Strong red fluorescence was observed in the Hut78 cells under the fluorescent microscope after co-transfection of the cells with the 3 plasmids of the lentiviral vector.The transfection efficiency of the collected virus exceeded 95％ in the Hut78 cells.The lentiviral vector significantly inhibited the expression of EZH2 at both the mRNA and protein levels.Conclusions The lentiviral RNAi vector of EZH2 has been successfully packaged and identified. Key words: Peripheral T-cell lymphomas;  Enhancer of zeste homolog 2;  Lentiviral vector;  RNA interference