Abstract
Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.
Highlights
Viral vectors are commonly used when high transduction efficiencies and/or high levels of foreign gene expression are required [1,2]
In the present work we investigated how PF6 and the cationiclipid based Lipofectamine 2000 (LF2000) reagent could be used for the delivery of DNA and RNA based Semliki Forest virus (SFV) expression vectors into eukaryotic cells and examined the effects of these transfections on subsequent viral infection
We studied the possibility of combining novel transfection reagent PF6, which has been previously shown to transfect both smaller and larger nucleic acid molecules, with SFV replicationcompetent and replicon vector technology
Summary
Viral vectors are commonly used when high transduction efficiencies and/or high levels of foreign gene expression are required [1,2]. Full-length viral vectors ( termed replication competent vectors) contain all the characteristic genetic elements of infectious viruses and generally have the ability to spread the infection from the original host cell. This property separates them from other viral and non-viral vectors and creates a potential biological threat to the host. In addition to its broad tropism, the SFV has several beneficial properties as a potential vector, including high expression levels of viral subgenomic (SG) mRNA synthesized in infected cells. Additional benefits of the SFV as a vector include its small genome, which can be modified with ease using corresponding cDNA clones, and the ability of the viral RNA to induce a productive infection [5]
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