Name: changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro
Uploaded: 2011-09-12T14:00:00
Description: Watch this Scientific Journal Video about Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro at JoVE.com

This work was supported by the Slovenian Research Agency (project J2-9770, infrastructural center IP-0510 and program P2-0249). This video represents supplementary material for the "Electroporation-based Technologies and Treatments" scientific workshop and postgraduate course, organized by the Faculty of Electrical Engineering at the University of Ljubljana, Slovenia. Authors thank also Du&scaron;a Hod&#x17E;i&#x10D; for kindly providing plasmid DNA.

1. Cell culture, plasmid and buffer preparation for the experiment
<ol>
 <li>In this experiment Chinese hamster ovary cells (CHO-K1) are used. Cells are grown in a nutrient mixture HAM-F12 (PAA) supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 400 &micro;l/l gentamicin (all from Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany), and 1 ml/l crystacilin (Pliva, Zagreb, Croatia). Cells are kept at 37&deg;C in a humidified 5% CO2 atmosphere in the incubator for 24h.</li>
 <li>Amplify plasmid pEGFP-N1...

<ol>
	<li>Rebersek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporator+with+automatic+change+of+electric+field+direction+improves+gene+electrotransfer+in-vitro.">Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.</a> Biomed Eng Online. 6, 25-25 (2007).</li><li>Trontelj, K., Rebersek, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip+electrode+chamber+for+small+volume+electroporation,+electrofusion,+and+gene+transfection.">Tip electrode chamber for small volume electroporation, electrofusion, and gene transfection.</a> 18, (2006).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-841 (1982).</li><li>Daud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+trial+of+interleukin-12+plasmid+electroporation+in+patients+with+metastatic+melanoma.">Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.</a> J Clin Oncol. 26, 5896-5903 (2008).</li><li>Pavlin, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+conductivity+of+a+suspension+of+permeabilized+cells:+a+theoretical+analysis.">Effective conductivity of a suspension of permeabilized cells: a theoretical analysis.</a> Biophys J. 85, 719-729 (2003).</li><li>Xie, T., Sun, L., Zhao, H., Fuchs, J., Tsong, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+mechanisms+of+electric+field-induced+DNA+transfection.+IV.+Effects+of+DNA+topology+on+cell+uptake+and+transfection+efficiency.">Study of mechanisms of electric field-induced DNA transfection. 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Effects of DNA topology on cell uptake and transfection efficiency.</a> Biophys J. 63, 1026-1031 (1992).</li><li>Rols, M., Delteil, C., Serin, G., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effects+on+electrotransfection+of+mammalian+cells.">Temperature effects on electrotransfection of mammalian cells.</a> Nucleic Acids Res. 22, 540-540 (1994).</li><li>Golzio, M., Teissie, J., Rols, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+synchronization+effect+on+mammalian+cell+permeabilization+and+gene+delivery+by+electric+field.">Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field.</a> Biochim Biophys Acta. 1563, 23-28 (2002).</li><li>Haberl, S., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Mg+ions+on+efficiency+of+gene+electrotransfer+and+on+cell+electropermeabilization.">Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization.</a> Bioelectrochemistry. 79, 265-271 (2010).</li><li>Rols, M., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+mammalian+cells+to+macromolecules:+control+by+pulse+duration.">Electropermeabilization of mammalian cells to macromolecules: control by pulse duration.</a> Biophys J. 75, 1415-1423 (1998).</li><li>Kanduser, M., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+gene+electrotransfer+using+high-and+low-voltage+pulses-An+in+vitro+study.">Mechanisms involved in gene electrotransfer using high-and low-voltage pulses-An in vitro study.</a> Bioelectrochemistry. 74, 265-271 (2009).</li><li>Pavlin, M., Flisar, K., Kanduser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+electrophoresis+in+gene+electrotransfer.">The role of electrophoresis in gene electrotransfer.</a> J Membr Biol. 236, 75-79 (2010).</li><li>Sersa, G., Cemazar, M., Semrov, D., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+electrode+orientation+improves+the+efficacy+of+electrochemotherapy+of+solid+tumors+in+mice.">Changing electrode orientation improves the efficacy of electrochemotherapy of solid tumors in mice.</a> Bioelectrochem Bioenerg. 39, 61-66 (1996).</li><li>Faurie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-mediated+gene+transfer+and+expression+are+controlled+by+the+life-time+of+DNA/membrane+complex+formation.">Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation.</a> J Gene Med. 12, 117-125 (2010).</li></ol>

Gene electrotransfer is a versatile biotechnology technique that enables transfer of DNA into cells by means of applying short, high voltage electric pulses 3 and represents a safer alternative to viral vectors due to its safety, efficacy and ease of application. Although today gene electrotransfer is widely used to transfect all types of cells and first phase I clinical trial using this method has been reported 4, the underlying mechanisms are still not completely understood. It is known, that appl...

<table border="1"><tbody><tr><td>Name of the reagent</td>
		<td>Company</td>
		<td>Catalogue number</td>
		<td>Comments</td></tr>
	<tr><td>HAM-F12</td>
		<td>PAA</td>
		<td>E15-016</td>
		<td>culture medium</td></tr>
	<tr><td>L-glutamine</td>
		<td>Sigma-Aldrich</td>
		<td>G7513</td>
		<td>&nbsp;</td></tr>
	<tr><td>fetal bovine serum</td>
		<td>PAA</td>
		<td>A15-151</td>
		<td>&nbsp;</td></tr>
	<tr><td>gentamicin</td>
		<td>Sigma-Aldrich</td>
		<td>G1397</td>
		<td>antibiotic</td></tr>
	<tr><td>crystacilin</td>
		<td>Pliva</td>
		<td>625110</td>
		<td>antibiotic</td></tr>
	<tr><td>pEGFP-N1</td>
		<td>Clontech Laboratories</td>
		<td>6085-1</td>
		<td>plasmid DNA</td></tr>
	<tr><td>HiSpeed Plasmid Maxi Kit</td>
		<td>Qiagen</td>
		<td>12662</td>
		<td>&nbsp;</td></tr>
	<tr><td>Na2HPO4</td>
		<td>Merck</td>
		<td>F640786 933</td>
		<td>&nbsp;</td></tr>
	<tr><td>NaH2PO4</td>
		<td>TKI Hrastnik</td>
		<td>0795</td>
		<td>&nbsp;</td></tr>
	<tr><td>MgCl2</td>
		<td>Sigma-Aldrich</td>
		<td>M-8266</td>
		<td>&nbsp;</td></tr>
	<tr><td>sucrose</td>
		<td>Sigma-Aldrich</td>
		<td>16104</td>
		<td>&nbsp;</td></tr>
	<tr><td>trypsin/EDTA solution</td>
		<td>Sigma-Aldrich</td>
		<td>T4174</td>
		<td>&nbsp;</td></tr>
	<tr><td>pipette tip</td>
		<td>Custom made</td>
		<td>&nbsp;</td>
		<td>&nbsp;</td></tr>
	<tr><td>electric pulse generator</td>
		<td>Custom made</td>
		<td>&nbsp;</td>
		<td>&nbsp;</td></tr>
	<tr><td>6 well plate</td>
		<td>TPP</td>
		<td>92406</td>
		<td>&nbsp;</td></tr>
	<tr><td>15 ml centrifuge tube</td>
		<td>TPP</td>
		<td>91015</td>
		<td>&nbsp;</td></tr>
	<tr><td>75 cm2 culture flask</td>
		<td>TPP</td>
		<td>90076</td>
		<td>&nbsp;</td></tr></tbody></table>

changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.

Watch this Scientific Journal Video about Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro at JoVE.com

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene transfection by electroporation is improved approximately two times when orientation of electric field is changed during pulse application, while cell viability is not affected. The increase in gene transfection is caused by the increase of the membrane area which is made competent for DNA entry into the cell.

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

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