Construction and identification of the lentiviral vector system expressing MDR1 small interference RNA / 中国耳鼻咽喉头颈外科

ABSTRACT

OBJECTIVE To construct the lentiviral vector system expressing MDR1 small interference RNA, and identify reversal efficiency of multidrug resistant phenotype in the human laryngeal cancer multidrug resistance cell lines (LSC-1/TAX). METHODS Three target sequences of oligonucleotides were selected according to MDR-1 gene sequence, the complementary DNA contained both sense and antisense strands were designed and synthesized. After the oligonucleotides were inserted into the plasmid expression system pLVTHM, the plasmid was cotransfected along with pCMV-dR8.74 and pMD2G into 293T cell lines to package lentiviral particles. Interference efficiency of the lentiviral vector system expressing MDR1 small interference RNA was determined by RT-PCR, real time PCR and Western Blot in the human laryngeal cancer multidrug resistance cell lines (LSC-1/TAX), drug resistance was measured by MTT assay after interference. RESULTS It was confirmed by digestion and sequencing that lentiviral vector had the correct structure and could express the GFP and siRNA. The functional titer of concentrated virus was more than 1?108TU/ml. The vectors expressing 3 target sequences can infect LSC-1/TAX, and the third vector has the best interference efficiency. CONCLUSION The lentiviral vector system expres-sing MDR1 siRNA has been constructed, which is necessary to reverse multidrug resistance phenotype in the human laryngeal cancer multidrug resistance cell lines