Limits...
Gene electrotransfer enhanced by nanosecond pulsed electric fields.

Guo S, Jackson DL, Burcus NI, Chen YJ, Xiao S, Heller R - Mol Ther Methods Clin Dev (2014)

Bottom Line: We demonstrated that the effect of nsPEFs on gene transfection was time restricted.The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters.We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

View Article: PubMed Central - PubMed

Affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University , Norfolk, Virginia, USA.

ABSTRACT
The impact of nanosecond pulsed electric fields (nsPEFs) on gene electrotransfer has not been clearly demonstrated in previous studies. This study was conducted to evaluate the influence of nsPEFs on the delivery of plasmids encoding luciferase or green fluorescent protein and subsequent expression in HACAT keratinocyte cells. Delivery was performed using millisecond electric pulses (msEPs) with or without nsPEFs. In contrast to reports in the literature, we discovered that gene expression was significantly increased up to 40-fold by applying nsPEFs to cells first followed by one msEP but not in the opposite order. We demonstrated that the effect of nsPEFs on gene transfection was time restricted. The enhancement of gene expression occurred by applying one msEP immediately after nsPEFs and reached the maximum at posttreatment 5 minutes, slightly decreased at 15 minutes and had a residual effect at 1 hour. It appears that nsPEFs play a role as an amplifier without changing the trend of gene expression kinetics due to msEPs. The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters. We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

No MeSH data available.


Kinetics of gene expression of HACAT cells after gene electrotransfer (GET). (a,b) Luciferase expression of HACAT cells after GET with gWIZ-Luc. Groups, Ctr: plasmid alone. nsPEFs: treated with nsPEFs alone. ms-50, 130: treated with 1 msEP applied electric field 50 V or 130 V. Comb-50, 130: treated with nsPEFs then 5 minutes later with 1 msEP applied electric field 50 V or 130 V. nsPEF Parameters: 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP parameters: 5 ms, 1 pulse, applied electric field as indicated. Error bars represent SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362372&req=5

fig4: Kinetics of gene expression of HACAT cells after gene electrotransfer (GET). (a,b) Luciferase expression of HACAT cells after GET with gWIZ-Luc. Groups, Ctr: plasmid alone. nsPEFs: treated with nsPEFs alone. ms-50, 130: treated with 1 msEP applied electric field 50 V or 130 V. Comb-50, 130: treated with nsPEFs then 5 minutes later with 1 msEP applied electric field 50 V or 130 V. nsPEF Parameters: 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP parameters: 5 ms, 1 pulse, applied electric field as indicated. Error bars represent SD.

Mentions: We further addressed if cells pretreated with nsPEFs could change the kinetics of gene expression by msEPs. Utilizing the IVIS Spectrum system, luciferase expression was evaluated at different time points with the same transfected cells. Two kinetic models of gene expression were found after GET with/without nsPEFs. Under parameters not causing cell death, in this case there were 5 ms of pulse duration and 50 V of applied electric field for 1 msEP with or without 60 ns of pulse duration, 24 KV/cm of electric field strength, 1 Hz of frequency, and 23 pulses for nsPEFs (Figure 4). Gene expression with 1 msEP alone or combined with nsPEFs was the highest at day 1 and rapidly dropped to the background level at day 4. Compared to 1 msEP alone, the nsPEFs enhanced gene expression about four times (Figure 4a). On the other hand, using parameters that cause death of majority of cells (Figure 1), applied electric field of 130 V for 1 msEP without change of other electrical parameters, gene expression reached the highest at day 1 and dropped in 8 days but did not drop to background levels even after 18 days after transfection either with 1 msEP alone or with nsPEFs. In contrast to 1 msEP alone, the nsPEFs enhanced gene expression 1.7–2.8 times at different time points (Figure 4b). If we consider more cell death for the combined pulses than that for 1 msEP alone, the increase of expression on a per cell basis could reach 15-fold at day 1. For both kinetic models of gene expression, nsPEFs plays a role as an amplifier without changing the trend of gene expression due to msEPs.


Gene electrotransfer enhanced by nanosecond pulsed electric fields.

Guo S, Jackson DL, Burcus NI, Chen YJ, Xiao S, Heller R - Mol Ther Methods Clin Dev (2014)

Kinetics of gene expression of HACAT cells after gene electrotransfer (GET). (a,b) Luciferase expression of HACAT cells after GET with gWIZ-Luc. Groups, Ctr: plasmid alone. nsPEFs: treated with nsPEFs alone. ms-50, 130: treated with 1 msEP applied electric field 50 V or 130 V. Comb-50, 130: treated with nsPEFs then 5 minutes later with 1 msEP applied electric field 50 V or 130 V. nsPEF Parameters: 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP parameters: 5 ms, 1 pulse, applied electric field as indicated. Error bars represent SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362372&req=5

fig4: Kinetics of gene expression of HACAT cells after gene electrotransfer (GET). (a,b) Luciferase expression of HACAT cells after GET with gWIZ-Luc. Groups, Ctr: plasmid alone. nsPEFs: treated with nsPEFs alone. ms-50, 130: treated with 1 msEP applied electric field 50 V or 130 V. Comb-50, 130: treated with nsPEFs then 5 minutes later with 1 msEP applied electric field 50 V or 130 V. nsPEF Parameters: 60 ns, 24 KV/cm, 1 Hz, and 23 pulses; msEP parameters: 5 ms, 1 pulse, applied electric field as indicated. Error bars represent SD.
Mentions: We further addressed if cells pretreated with nsPEFs could change the kinetics of gene expression by msEPs. Utilizing the IVIS Spectrum system, luciferase expression was evaluated at different time points with the same transfected cells. Two kinetic models of gene expression were found after GET with/without nsPEFs. Under parameters not causing cell death, in this case there were 5 ms of pulse duration and 50 V of applied electric field for 1 msEP with or without 60 ns of pulse duration, 24 KV/cm of electric field strength, 1 Hz of frequency, and 23 pulses for nsPEFs (Figure 4). Gene expression with 1 msEP alone or combined with nsPEFs was the highest at day 1 and rapidly dropped to the background level at day 4. Compared to 1 msEP alone, the nsPEFs enhanced gene expression about four times (Figure 4a). On the other hand, using parameters that cause death of majority of cells (Figure 1), applied electric field of 130 V for 1 msEP without change of other electrical parameters, gene expression reached the highest at day 1 and dropped in 8 days but did not drop to background levels even after 18 days after transfection either with 1 msEP alone or with nsPEFs. In contrast to 1 msEP alone, the nsPEFs enhanced gene expression 1.7–2.8 times at different time points (Figure 4b). If we consider more cell death for the combined pulses than that for 1 msEP alone, the increase of expression on a per cell basis could reach 15-fold at day 1. For both kinetic models of gene expression, nsPEFs plays a role as an amplifier without changing the trend of gene expression due to msEPs.

Bottom Line: We demonstrated that the effect of nsPEFs on gene transfection was time restricted.The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters.We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

View Article: PubMed Central - PubMed

Affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University , Norfolk, Virginia, USA.

ABSTRACT
The impact of nanosecond pulsed electric fields (nsPEFs) on gene electrotransfer has not been clearly demonstrated in previous studies. This study was conducted to evaluate the influence of nsPEFs on the delivery of plasmids encoding luciferase or green fluorescent protein and subsequent expression in HACAT keratinocyte cells. Delivery was performed using millisecond electric pulses (msEPs) with or without nsPEFs. In contrast to reports in the literature, we discovered that gene expression was significantly increased up to 40-fold by applying nsPEFs to cells first followed by one msEP but not in the opposite order. We demonstrated that the effect of nsPEFs on gene transfection was time restricted. The enhancement of gene expression occurred by applying one msEP immediately after nsPEFs and reached the maximum at posttreatment 5 minutes, slightly decreased at 15 minutes and had a residual effect at 1 hour. It appears that nsPEFs play a role as an amplifier without changing the trend of gene expression kinetics due to msEPs. The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters. We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer.

No MeSH data available.