Production of Recombinant Lentiviruses Expressing miR-۱۶ by Transient Transfection of ۲۹۳T Cells

Objective: The aim of the present study was the production of recombinant lentviruses that express miR-۱۶. After transduction, altered expression levels of miRNA and its target protein were analyzed. Methods: A DNA fragment that contained the miR-۱۶ precursor was cloned in a lentiviral plasmid. Lentiviral vector particles were produced by transient calcium phosphate co-transfection of ۲۹۳T 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-۱۶ 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 ۲۹۳T 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 ۸۰% of the cells transduced with lentivirus at MOI=۱. Real-time PCR assay showed that miR-۱۶ expression levels significantly increased in transduced cells compared with the control group. As shown by Western blot analysis, miR-۱۶ overexpression downregulated Bcl-۲ expression at the protein level. Conclusion: This lentivirus expression system could be considered as a tool for efficient delivery of produced miRNAs to cells.

کلیدواژه ها:

Transfection ، microRNA ، Lentivirus ، Transduction ، ترانسفکشن ، میکرو RNA ، لنتیویروس ، ترانسداکشن