title: A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy.
abstract: Suicide gene therapy is a therapeutic strategy, in which cell suicide inducing transgenes are introduced into target cells. Inserting a toxin-encoding gene into a lentiviral vector leads to decreased efficiency of virus production due to lethal effect of toxin on packaging cells. In this study, we designed and constructed a transfer vector to express the toxin in transduced cells but not in packaging cells. Plasmid pLenti-F/GFP was constructed by cutting out R 5'LTR-R 3'LTR fragment with the AflII restriction endonuclease from a plasmid pLenti4-GW/H1/TO-laminshRNA, followed by ligating R 5'LTR-R 3'LTR fragment, constructed by three PCR stages. The promoter and GFP CDS were inserted in opposite strand. For lentiviral production, the HEK293T cell line was co-transfected with the PMD2G, psPAX2, and pLenti-F/GFP plasmids (envelope, packaging, and transfer plasmids).Viral vector titers were assayed. The HEK293T cell line was transduced with this virus. PCR was performed to confirm the presence of the promoter fragment between the R and U5 in 3'LTR. The lentivirus titers were approximately 2 × 10(5). The GFP expression was seen in 51 % of the HEK293T cells transduced with lentivirus. The PCR product size was 1440 bp confirming the promoter fragment position between the R and U5 in 3'LTR. The strategy enables us to use a broad spectrum of toxin genes in gene therapy and helps avoid the death of the packaging cells with lentiviral vectors carrying a toxin-encoding gene, thereby increasing the efficiency of viral production in packaging cells.
authors.affiliation: Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Mohammadi
authors.forename: Laleh
authors.initials: L
authors.lastname: Shariati
authors.forename: Hossein
authors.initials: H
authors.lastname: Khanahmad
authors.forename: Mahsa
authors.initials: M
authors.lastname: Kolahdouz
authors.forename: Fariborz
authors.initials: F
authors.lastname: Kianpoor
authors.forename: Jahan Afrooz
authors.initials: JA
authors.lastname: Ghanbari
authors.forename: Zahra
authors.initials: Z
authors.lastname: Hejazi
authors.forename: Mansoor
authors.initials: M
authors.lastname: Salehi
authors.forename: Parvaneh
authors.initials: P
authors.lastname: Nikpour
authors.forename: Mohammad Amin
authors.initials: MA
authors.lastname: Tabatabaiefar
mesh_terms: D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*