Golestan University of Medical Sciences Repository

Use of integrase-minus lentiviral vector for transient expression

Farazmandfar, T. and Shahreza, H.K. and Haghshenas, M.R. and Janbabai, G. and Azadeh, H. and Samaei, N.M. (2012) Use of integrase-minus lentiviral vector for transient expression. Cell Journal, 14 (2). pp. 76-81. ISSN 22285806 (ISSN)

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Abstract

Objective: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector. Materials and Methods: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP) gene expression by a fluorescence microscopy. Results: Recombinant and wild lentiviruses titer was about 5�8�10 6 transducing units/ ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed. Conclusion: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.

Item Type: Article
Additional Information: Unmapped bibliographic data: LA - English [Field not mapped to EPrints] J2 - Cell J. [Field not mapped to EPrints] AD - Faculty of Advanced Medical Science Technology, Golestan University of Medical Sciences, Gorgan, Iran [Field not mapped to EPrints] AD - Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran [Field not mapped to EPrints] AD - Cancer Research Center, Mazandaran University of Medical Sciences, Sari, Iran [Field not mapped to EPrints] AD - Pasteur Research and Production Complex, Karaj, Iran [Field not mapped to EPrints] DB - Scopus [Field not mapped to EPrints]
Uncontrolled Keywords: Integrase-Minus, Lentiviral Vector, Transient Expression, alanine, aspartic acid, glutamic acid, green fluorescent protein, integrase, integrase minus lentiviral vector, lentivirus vector, unclassified drug, valine, amino acid sequence, animal cell, article, controlled study, enzyme active site, fluorescence microscopy, gene cassette, gene structure, gene targeting, genetic transfection, missense mutation, nonhuman, site directed mutagenesis, transient expression, viral gene delivery system, virus genome
Subjects: مقالات نمایه شده محققین دانشگاه در سایت ,Web of Science ,Scopus
موارد کلی
Divisions: معاونت تحقیقات و فناوری
Depositing User: GOUMS
Date Deposited: 15 Apr 2015 10:23
Last Modified: 11 May 2015 15:03
URI: http://eprints.goums.ac.ir/id/eprint/1999

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