Babashah, Sadegh, Sadeghizadeh, Majid, Soleimani, Masoud, Sadeghizadeh, Majid, Hajifathali, Abbas, Rezaei Tavirani, Mostafa (2012). Production of Recombinant Lentiviruses Expressing miR-16 by Transient Transfection of 293T Cells. , 15(1), 1-12.
Sadegh Babashah; Majid Sadeghizadeh; Masoud Soleimani; Majid Sadeghizadeh; Abbas Hajifathali; Mostafa Rezaei Tavirani. "Production of Recombinant Lentiviruses Expressing miR-16 by Transient Transfection of 293T Cells". , 15, 1, 2012, 1-12.
Babashah, Sadegh, Sadeghizadeh, Majid, Soleimani, Masoud, Sadeghizadeh, Majid, Hajifathali, Abbas, Rezaei Tavirani, Mostafa (2012). 'Production of Recombinant Lentiviruses Expressing miR-16 by Transient Transfection of 293T Cells', , 15(1), pp. 1-12.
Babashah, Sadegh, Sadeghizadeh, Majid, Soleimani, Masoud, Sadeghizadeh, Majid, Hajifathali, Abbas, Rezaei Tavirani, Mostafa Production of Recombinant Lentiviruses Expressing miR-16 by Transient Transfection of 293T Cells. , 2012; 15(1): 1-12.

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

Pathobiology Reserach
Article 1, Volume 15, Issue 1, 2012, Pages 1-12    PDF (1.57 M)
Authors
Sadegh Babashah1; Majid Sadeghizadeh* 1; Masoud Soleimani2; Majid Sadeghizadeh* 1; Abbas Hajifathali3; Mostafa Rezaei Tavirani4
1Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
2Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
3Taleghani Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4Proteomics 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
References
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