Iranian Biomedical Journal
مجله بیومدیکال ایران
IBJ
Basic Sciences
http://ibj.pasteur.ac.ir
1
admin
1028-852X
2008-823X
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10.61186/ibj
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8888
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en
jalali
1396
4
1
gregorian
2017
7
1
21
4
online
1
fulltext
en
Construction of Various γ34.5 Deleted Fluorescent-Expressing Oncolytic herpes Simplex type 1 (oHSV) for Generation and Isolation of HSV-Based Vectors
Medical Biotechnology
Medical Biotechnology
مقاله کامل
Full Length/Original Article
<p><strong>Background:</strong> Oncolytic herpes simplex virus (oHSV)-based vectors lacking <em>γ34.5 </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>γ34.5</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<sup>nd</sup> and 3<sup>rd</sup> 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>
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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
100319475328460055526
100319475328460055526
Yes
Department of Virology, Pasteur Institute of Iran, Tehran, Iran
Farzin
Roohvand
100319475328460055527
100319475328460055527
No
Department of Virology, Pasteur Institute of Iran, Tehran, Iran
Ladan
Teimoori-Toolabi
100319475328460055528
100319475328460055528
No
Molecular Medicine Department, Biotechnology Research center, Pasteur institute of Iran, Tehran, Iran
Mohammad Ali
Shokrgozar
100319475328460055529
100319475328460055529
No
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
Mina
Bahrololoumi
100319475328460055530
100319475328460055530
No
Department of Virology, Pasteur Institute of Iran, Tehran, Iran
Kayhan
Azadmanesh
100319475328460055531
100319475328460055531
No
Department of Virology, Pasteur Institute of Iran, Tehran, Iran