A preliminary step of a novel strategy in suicide gene therapy with lentiviral vector
نویسندگان
1 Department of Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Microbiology, Arak University of Medical Sciences, Arak, Iran
3 Physiology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
4 Department of Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran
5 Department of Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran
6 Department of Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran
7 Department of Biostatistics of Public Health, Isfahan University of Medical Sciences, Isfahan, Iran
8 MS in Botanical Biology, Department of Biology, Payamnoor University, Isfahan, Iran
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چکیده
Background: One of the challenges in lentiviral vector-based suicide gene therapy by toxin or apoptosis-inducing genes is death of packaging cells. Therefore, the process of production of these lentiviral particles would be stopped in this step. We proposed that insertion of a reverse promoter between R and U5 regions of 5' long terminal repeat (LTR) in transfer plasmid could be considered as a solution for this problem. But it is not known, whether the insertion of RΔU3 sequence between the promoter and target gene in proviral genome during the life-cycle of lentivirus may interfere whit gene expression in target cells.Materials and Methods: The following were performed in this study: insertion of RΔU3 sequence in pEGFP-N1 plasmid, evaluation of the expression of eGFP gene after calcium phosphate co-precipitation transfection of pCMV-RΔU3-GFP construction in 293T cells, and quantitative assay of eGFP gene by flow cytometry technique.Results: Our results from flow cytometry technique analysis showed that there was no significant difference between the expression of eGFP gene in transfected cells with pEGFP-N1 and pCMV-RΔU3-GFP plasmids (P > 0.05).Conclusion: In this step of our strategy, we demonstrated that modification of orientation and location of promoter may overcome some issues in lentiviral suicide gene therapy, especially when toxin or apoptosis-inducing genes are used.
