production of recombinant lentiviruses expressing mir-16 by transient transfection of 293t cells

objective: the aim of the present study was the production of recombinant lentviruses that express mir-16. after transduction, altered expression levels of mirna and its target protein were analyzed. methods: a dna fragment that contained the mir-16 precursor was cloned in a lentiviral plasmid. lentiviral vector particles were produced by transient calcium phosphate co-transfection of 293t cells with the combined lenti-mir, structural and packaging plasmids. viral supernatants were harvested and concentrated by ultracentrifuge. virus titration was determined by fluorescent microscopy and flow cytometry. altered expression levels of mir-16 were evaluated by real-time pcr; its protein target was evaluated by western blot. results: the identity of dna was established by colony-pcr, enzymatic digestion of positive clones, and dna sequencing. after co-transfection of 293t cells with the combined lenti-mir, structural and packaging plasmids, viral particles were concentrated and the virus titer determined. maximum expression of the gfp reporter gene was obtained in more than 80% of the cells transduced with lentivirus at moi=1. real-time pcr assay showed that mir-16 expression levels significantly increased in transduced cells compared with the control group. as shown by western blot analysis, mir-16 overexpression downregulated bcl-2 expression at the protein level. conclusion: this lentivirus expression system could be considered as a tool for efficient delivery of produced mirnas to cells.