title: Use of integrase-minus lentiviral vector for transient expression.
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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 1. Faculty of Advanced Medical Science Technology, Golestan University of Medical Sciences, Gorgan, Iran ; 2. Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran ; 3. Cancer Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
authors.forename: Touraj
authors.initials: T
authors.lastname: Farazmandfar
authors.forename: Hossein
authors.initials: H
authors.lastname: Khanahmad Shahreza
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Haghshenas
authors.forename: Ghasem
authors.initials: G
authors.lastname: Janbabai
authors.forename: Hossein
authors.initials: H
authors.lastname: Azadeh
authors.forename: Nader
authors.initials: N
authors.lastname: Mansour Samaei
keywords: Integrase-Minus; Lentiviral Vector; Transient Expression