title: [Establishment of a U266 cell line with stable Bmi-1 silencing by lentivirus-mediated RNA interference].
abstract: 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.
authors.affiliation: Fujian Institute of Hematology, Union Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
authors.forename: Zhen-Zhen
authors.initials: ZZ
authors.lastname: Xu
authors.forename: Shun-Quan
authors.initials: SQ
authors.lastname: Wu
authors.forename: Rong
authors.initials: R
authors.lastname: Zhan
mesh_terms: D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D063150:Polycomb Repressive Complex 1 / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: C069077:BMI1 protein, human; D034741:RNA, Small Interfering; D063150:Polycomb Repressive Complex 1