Production of Recombinant Lentiviruses Expressing miR-16 by Transient Transfection of 293T Cells

Authors
Sadegh Babashah 1
Majid Sadeghizadeh 1
Masoud Soleimani 2
Majid Sadeghizadeh 1
Abbas Hajifathali 3
Mostafa Rezaei Tavirani 4
1 Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
2 Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
3 Taleghani Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Proteomics Research Center, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract
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.
Keywords
Transfection
microRNA
Lentivirus
Transduction
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Pathobiology Research
Volume 15, Issue 1
Spring 2012
Pages 1-12