Document Type : مقاله‌های پژوهشی

Abstract

Introduction: Biological methods or viral vectors are used extensively for gene transfer for therapeutic aims. Lentivial vectors, with advantages such as the capacity of transducing dividing and non-dividing cells, carrying large genetic payloads and stable long-term transgene expression, are considered suitable tools for gene transfer for research and therapeutic purposes. The aim of this study was to produce a lentiviral vector based on HIV-1, with vesicular stomatitis virus glycoprotein-G: VSVG. This vector can be used as a tool for gene transfer to dividing and non-dividing cells for research and therapeutic purposes. Materials and methods: Calcium phosphate method was used for co-transfection of three plasmids pWPXL-GFP (expression vector with reporter gene), psPAX2 consisting of gag, pol gene of HIV-1 virus and pMD2.G (Envelope vector) on HEK293T human embryo cell line. Flow cytometry technique was used for assessment of green fluorescent protein (GFP) expression as a reporter gene. Viruses were concentrated and used for transduction on the target cell line. Results: GFP expression was observed in 51.37% of transfected HEK293T cell line. After concentrating the viruses and transduction, pro-virus penetration into the target cell genome was observed with long-term green positive expression in transduced cells using fluorescent microscopy 15 days after transduction. Conclusion: The ability of constructed viruses to enter host cells was confirmed by GFP expression in these cells. The lentivirus (and therefore the vector) produced in this study proved effective in transducing dividing and non-dividing cells, making it more efficient than other viral vectors in gene transfer investigations.   Key words: Calcium phosphate, Gene transfer, Lentiviral vectors, Transduction, Transfection