PRODUCTION OF RECOMBINANT LENTIVIRUSES EXPRESSING MIR-16 BY TRANSIENT TRANSFECTION OF 293T CELLS

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BABASHAH SADEGH; SADEGHIZADEH MAJID; SOLEIMANI MASOUD; HAJIFATHALI ABBAS; REZAEI TAVIRANI MOSTAFA

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Journal: Pathobiology Research Year:2012 | Volume:15 | Issue:1 Page(s): 1-12

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Title

PRODUCTION OF RECOMBINANT LENTIVIRUSES EXPRESSING MIR-16 BY TRANSIENT TRANSFECTION OF 293T CELLS

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Keywords

MICRORNAQ2

LENTIVIRUSQ2

TRANSFECTIONQ3

TRANSDUCTIONQ2

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 down regulated 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.

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APA: Copy

BABASHAH, SADEGH, SADEGHIZADEH, MAJID, SOLEIMANI, MASOUD, HAJIFATHALI, ABBAS, & REZAEI TAVIRANI, MOSTAFA. (2012). PRODUCTION OF RECOMBINANT LENTIVIRUSES EXPRESSING MIR-16 BY TRANSIENT TRANSFECTION OF 293T CELLS. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), 15(1), 1-12. SID. https://sid.ir/paper/80986/en

Vancouver: Copy

BABASHAH SADEGH, SADEGHIZADEH MAJID, SOLEIMANI MASOUD, HAJIFATHALI ABBAS, REZAEI TAVIRANI MOSTAFA. PRODUCTION OF RECOMBINANT LENTIVIRUSES EXPRESSING MIR-16 BY TRANSIENT TRANSFECTION OF 293T CELLS. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES)[Internet]. 2012;15(1):1-12. Available from: https://sid.ir/paper/80986/en

IEEE: Copy

SADEGH BABASHAH, MAJID SADEGHIZADEH, MASOUD SOLEIMANI, ABBAS HAJIFATHALI, and MOSTAFA REZAEI TAVIRANI, “PRODUCTION OF RECOMBINANT LENTIVIRUSES EXPRESSING MIR-16 BY TRANSIENT TRANSFECTION OF 293T CELLS,” PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), vol. 15, no. 1, pp. 1–12, 2012, [Online]. Available: https://sid.ir/paper/80986/en

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