A comparative investigation on efficiency of bacteriophage lambda and M13 based vectors for delivering and expression of transgene in eukaryote cells

Abstract

Gene delivery might be affected by several tribulations based on carrier/vector applied. Bacteriophages lambda and M13 have different genome conformations; linear double-stranded and circular single-stranded respectively. Therefore, it might be expected that these two common classes of gene delivery vehicles will have different capacity for gene delivery and expression in eukaryote cells. To address the possible effects of linear double-stranded and circular single-stranded genome conformations of bacteriophages lambda and M13 on the transgene expression, the transfection efficacy of two vectors based on lambda and M13 were compared in AGS cell line. The GFP encoding sequence was inserted into the Lambda ZAP-CMV XR vector which resulted in λ-ZAP-CMV-GFP construct. The construct was then in vitro packaged using Gigapack® III Gold packaging extract and λ-GFP phage particles were obtained. The λ-GFP phage particles were then used for in vivo excisioning which resulted in M13-CMV-Script-GFP construct. 1011 copy of λ-ZAP-CMV-GFP or M13-CMV-Script-GFP constructs were transfected into AGS cells using lipofectamine 2000. Transfection efficiencies were analyzed by FACS. Results showed that linear double-stranded λ-ZAP-CMV-GFP was efficient than single-stranded form of M13-CMV-Script-GFP while its double-stranded form was efficient than the linear double-stranded λ-ZAP-CMV-GFP construct for transgene delivery and expression. Moreover the GFP signals resulted from transfections by single-stranded form of M13-CMV-Script-GFP construct faded more quickly in comparison to others. These findings highlight that genome conformation of gene carriers might be an important factor when seeking for an appropriate gene carrier/vehicle.

Keywords

• gene delivery
• phage-mediated gene transfection
• vector conformation