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

Summary

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.

Abstract

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.

Protocol

1. Cell culture, plasmid and buffer preparation for the experiment

1. 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 µl/l gentamicin (all from Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany), and 1 ml/l crystacilin (Pliva, Zagreb, Croatia). Cells are kept at 37°C in a humidified 5% CO₂ atmosphere in the incubator for 24h.
2. Amplify plasmid pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, USA) encoding green fluorescent protein (GFP) in DH5α strain of Escherichia coli and isolate it with HiSpeed Pl....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HAM-F12	PAA Laboratories	E15-016	culture medium
L-glutamine	Sigma-Aldrich	G7513
fetal bovine serum	PAA Laboratories	A15-151
gentamicin	Sigma-Aldrich	G1397	antibiotic
crystacilin	Pliva	625110	antibiotic
pEGFP-N1	Clontech Laboratories	6085-1	plasmid DNA
HiSpeed Plasmid Maxi Kit	Qiagen	12662
Na2HPO4	Merck & Co., Inc.	F640786 933
NaH2PO4	TKI Hrastnik	0795
MgCl2	Sigma-Aldrich	M-8266
sucrose	Sigma-Aldrich	16104
trypsin/EDTA solution	Sigma-Aldrich	T4174
pipette tip	Custom Made
electric pulse generator	Custom Made
6 well plate	Techno Plastic Products	92406
15 ml centrifuge tube	Techno Plastic Products	91015
75 cm2 culture flask	Techno Plastic Products	90076