en Medical Biotechnology Medical Biotechnology مقاله کامل Full Length/Original Article <p><strong>Background:</strong> Oncolytic herpes simplex virus (oHSV)-based vectors lacking <em>&gamma;34.5 </em>gene<em>,</em> are considered as ideal templates to construct efficient vectors for (targeted) cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and <em>&gamma;34.5</em>-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors. <strong>Methods:</strong> Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein (GFP) and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines. <strong>Results:</strong> We generated three recombinant viruses, HSV-GFP, HSV-GR (Green-Red), and HSV-Red. The HSV-GFP showed two log higher titer (1010 PFU) than wild type (108 PFU). In contrast, HSV-GR and HSV-Red showed one log lower titer (107 PFU) than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 (<em>P</em><0.001). Moreover, HSV-GFP showed higher infection potency (98%) in comparison with HSV-GR (82%). <strong>Conclusion:</strong> Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2^nd and 3^rd generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells <em>in vitro</em>/<em>in vivo</em> and as oncolytic agents in cancer therapy.</p> <div></div> Oncolytic viruses, Herpes simplex virus, Homologous recombination, Flow cytometry, Fluorescence microscopy 206 217 http://ibj.pasteur.ac.ir/browse.php?a_code=A-10-1-661&slc_lang=en&sid=1 Shahriyar Abdoli Yes Department of Virology, Pasteur Institute of Iran, Tehran, Iran Farzin Roohvand No Department of Virology, Pasteur Institute of Iran, Tehran, Iran Ladan Teimoori-Toolabi No Molecular Medicine Department, Biotechnology Research center, Pasteur institute of Iran, Tehran, Iran Mohammad Ali Shokrgozar No National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran Mina Bahrololoumi No Department of Virology, Pasteur Institute of Iran, Tehran, Iran Kayhan Azadmanesh No Department of Virology, Pasteur Institute of Iran, Tehran, Iran