Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System

Microbial Cell Factories

Research

Nafiseh Nafissi¹  Roderick Slavcev¹ 

[1] School of Pharmacy, Faculty of Science, University of Waterloo, N2L 3G1, Waterloo, Ontario, Canada;

关键词: Mini DNA vectors;    Linear covalently closed plasmid vector;    DNA vector integration;    Non-viral gene delivery;    “minicircles”;    pal;    telRL;    loxP;    Bacterial engineering;   

DOI  :  10.1186/1475-2859-11-154

 received in 2012-10-02, accepted in 2012-11-25,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundWhile safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event.ResultsWe constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called “Super Sequence”, and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc—linear covalently closed (Tel/TelN-cell), or mini ccc—circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpart.ConclusionWe offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of “minicircle” DNA vectors and virtually eliminate the potential for undesirable vector integration events.

【 授权许可】

CC BY   
© Nafissi and Slavcev; licensee BioMed Central Ltd. 2012