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Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro
Published on: September 12, 2011
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Gene electrotransfer enhanced by nanosecond pulsed electric fields.
Siqi Guo1, Diane L Jackson1, Niculina I Burcus1
1Frank Reidy Research Center for Bioelectrics, Old Dominion University , Norfolk, Virginia, USA.
Molecular Therapy. Methods & Clinical Development
|May 28, 2015
Summary
Nanosecond pulsed electric fields (nsPEFs) significantly enhance gene electrotransfer when applied before millisecond electric pulses (msEPs), boosting gene expression up to 40-fold in keratinocytes. This nsPEF amplification effect is time-sensitive and involves calcium-dependent and independent pathways.
Area of Science:
- Biophysics
- Molecular Biology
- Cell Biology
Background:
- Gene electrotransfer is a method for delivering nucleic acids into cells.
- The role of nanosecond pulsed electric fields (nsPEFs) in enhancing gene electrotransfer requires further clarification.
- Previous studies have not clearly demonstrated the impact of nsPEFs on gene electrotransfer efficiency.
Purpose of the Study:
- To investigate the influence of nsPEFs on gene delivery and expression in HACAT keratinocyte cells.
- To evaluate the combined effects of nsPEFs and millisecond electric pulses (msEPs) on gene electrotransfer.
- To determine the optimal order and timing of nsPEF and msEP application for enhanced gene expression.
Main Methods:
- Gene delivery of luciferase and green fluorescent protein plasmids into HACAT cells.
- Application of millisecond electric pulses (msEPs) with or without preceding nsPEFs.
- Assessment of gene expression levels and cell viability.
- Analysis of the temporal effects of nsPEFs on gene electrotransfer.
Main Results:
- Applying nsPEFs before a single msEP significantly increased gene expression up to 40-fold, contrary to previous reports.
- The order of pulse application was critical; nsPEFs followed by msEPs enhanced expression, but not vice versa.
- The enhancing effect of nsPEFs was time-restricted, with maximum enhancement observed immediately after nsPEF application and a residual effect up to 1 hour.
- nsPEFs acted as an amplifier of gene expression kinetics induced by msEPs.
- Cell viability was dependent on specific nsPEF parameters.
- Both calcium-dependent and independent mechanisms were implicated in the nsPEF-mediated enhancement of gene electrotransfer.
Conclusions:
- nsPEFs can significantly amplify gene electrotransfer efficiency when applied prior to msEPs in keratinocytes.
- The temporal dynamics and order of nsPEF application are crucial for maximizing gene delivery and expression.
- Understanding the underlying calcium-dependent and independent mechanisms can inform the development of improved gene electrotransfer protocols.
