Construction of pSiRNA-Notch1 recombinant retroviral vector and its effect on malignant glioma cells in vitro / 第三军医大学学报

ABSTRACT

Objective To construct a retrovirus-mediated expression system containing double strands DNA for RNA interference on human Notch1 gene and study its inhibitory effect on human glioma cell lines U251 and CHG-5 in vitro.Methods A recombinant retroviral vector pSiRNA-Notch1 was generated by cloning a double strands DNA for RNA interference on human Notch1 gene into a retroviral vector Psilencer 5.1-H1 Retro.Human glioma cell lines U251 and CHG-5 were infected with the viral supernatant from the PT67 clones.After 3 d,the viability,Notch1 mRNA and protein of the transfected cells were examined by WST-8 assay,RT-PCR and Western blot.Results The pSiRNA-Notch1 recombinant retroviral vector had been constructed correctly.The titer assayed on NIH3T3 cells was up to 224?104 cfu/ml.Three days after transfection,the viability,Notch1 mRNA and protein of the transfected cells decreased significantly.Conclusion The constructed pSiRNA-Notch1 retroviral vector shows effective inhibition effect on the viability in human malignant glioma cells,with potential utility in the gene therapy for human malignant glioma.