Construction and identification of lentiviral vector of RNA interference of E2F-1 gene

Objective: To construct a lentiviral vector-mediated RNA interference(RNAi) of targeting gene E2F-1.Methods: Two short hairpin RNA oligonucleotides targeting gene E2F-1 on appropriate site and one pair of negative control oligonucleotide sequence were synthesized and inserted into pLKO.1-PURO-U6(Age I/EcoR I) vector.The sequences of three plasmids were identified by DNA sequencer and restriction endonuclease digestion.These recombinant plasmids were transfected into the 293T cells along with lentiviral packing mix by lipofectamine 2000 for the package of lentiviral particles.Then the lentiviral vector particles were transfected into Tca8113 cells and E2F-1 expression in the transfected cells was assayed by Real-Time PCR and Western.Results: It was verified that the specific DNA oligonucleotide was cloned into the vector successfully.In the first group,the expression of E2F-1 mRNA and the expression of E2F-1 protein in Tca8113 cells was significantly reduced after transfecting the recombinant plasmids,compared to the control.Conclusions: It indicates the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of E2F-1 mRNA,and those provides the precondition for the further study of E2F-1 in OSCC(oral squamous cell carcinoma) pathogenesis by using lentivirus system to construct stably silencing E2F-1 in OSCC cell lines.