[Establishment of a U266 cell line with stable Bmi-1 silencing by lentivirus-mediated RNA interference]

This study was aimed to construct lentivirus-mediated shRNA expression vector targeting Bmi-1 and establish a stable cell line U266-li, so as to pave the way for further research on function of Bmi-1 and application of shRNA to gene therapy. One pair of oligonucleotide sequences targeted at human Bmi-1 mRNA were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pLVTHM vector. Virus particles were collected after the control or shRNA vectors were co-transfected with the psPAX2 packaging plasmid and the plasmid pMD2.G was enveloped into HEK-293T cells by using Lipofectamine2000. The U266 cells were transduced with 5 × 10(6) recombinant lentivirus-transducing units plus 6 µg/ml of polybrene. Real-time PCR and Western blot were used respectively to detect the expression of Bmi-1 and P14 after lentivirus transduction. DNA sequencing demonstrated that the lentivirus RNAi vector of Bmi-1 was constructed successfully and the virus was packaged in 293T cells. The titer of virus was 5 × 10(7) TU/ml. Stable transfected U266 cell line was established. As was expected, the mRNA and protein levels of Bmi-1 was reduced significantly in U266 cells after lentivirus transduction, whereas the mRNA and protein levels of P14 was upregulated. It is concluded that the lentiviral RNAi vector of Bmi-1 is constructed, and U266 stable cell line is established.