Effects of pulse strength and pulse duration on in vitro DNA electromobility.
Interstitial transport of DNA is a rate-limiting step in electric field-mediated gene delivery in vivo. Interstitial transport of macromolecules, such as plasmid DNA, over a distance of several cell layers, is inefficient due to small diffusion coefficient and inadequate convection. Therefore, we explored electric field as a novel driving force for interstitial transport of plasmid DNA. In this study, agarose gels were used to mimic the interstitium in tissues as they had been well characterized and could be prepared reproducibly. We measured the electrophoretic movements of fluorescently labeled plasmid DNA in agarose gels with three different concentrations (1.0%, 2.0% and 3.0%) subjected to electric pulses at three different field strengths (100, 200 and 400 V/cm) and four different pulse durations (10, 50, 75, 99 ms). We observed that: (1) shorter pulses (10 ms) were not as efficient as longer pulses in facilitating plasmid transport through agarose gels; (2) plasmid electromobility reached a plateau at longer pulse durations; and (3) plasmid electromobility increased with applied electric energy, up to a threshold, in all three gels. These data suggested that both pulse strength and duration needed to be adequately high for efficient plasmid transport through extracellular matrix. We also found that electric field was better than concentration gradient of DNA as a driving force for interstitial transport of plasmid DNA.
Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Durham, NC 27708, USA.
David A
DA
Zaharoff
Fan
F
Yuan
D004247:DNA / Q000737:chemistry*; D004563:Electrochemistry; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D005453:Fluorescence; D005782:Gels / Q000737:chemistry; D018014:Gene Transfer Techniques; D066298:In Vitro Techniques; D007477:Ions / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000235:genetics
D005782:Gels; D007477:Ions; D004247:DNA