Transfection by Electroporation

Huntington Potter1, Richard Heller2

1 Byrd Alzheimer's Institute, University of South Florida College of Medicine, Tampa, Florida, 2 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Appendix 1E
DOI:  10.1002/0471142301.nsa01es57
Online Posting Date:  October, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Electroporation—the use of high‐voltage electric shocks to introduce DNA into cells—can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knock‐out, knock‐in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination. Also described are modifications for preparation and transfection of plant protoplasts. Curr. Protoc. Neurosci. 57:A.1E.1‐A.1E.11. © 2011 by John Wiley & Sons, Inc.

Keywords: molecular biology; introduction of DNA into cells; gene regulation; gene expression; transcription and translation; gene therapy; DNA vaccine

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Electroporation into Mammalian Cells
  • Basic Protocol 2: Electroporation into Muscle or Skin
  • Alternate Protocol 1: Electroporation into Plant Protoplasts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Electroporation into Mammalian Cells

  Materials
  • Mammalian cells to be transfected
  • Complete medium ( appendix 2A) without and with appropriate selective agents (unit 4.6)
  • Electroporation buffer (see recipe), ice‐cold
  • Linear or supercoiled, purified DNA preparation (see step 7)
  • Centrifuge with Beckman JS‐4.2 rotor or equivalent
  • Electroporation cuvettes (Bio‐Rad, cat. no. 165‐2088)
  • Electroporator (Bio‐Rad Gene Pulser X‐Cell, or equivalent) and power source
  • Additional reagents and equipment for stable transformation in selective medium (unit 4.6)

Basic Protocol 2: Electroporation into Muscle or Skin

  Materials
  • Linear or supercoiled, purified DNA preparation (see annotation to step 1)
  • DNA amplification kit (e.g., Qiagen, cat. no. 12991)
  • Animals to undergo procedure
  • Anesthetic: 2% to 4% isoflurane in O 2
  • Electric razor, disposable razor, or hair removal product
  • Anesthesia apparatus
  • 1‐ml syringe
  • 25‐ to 30‐G needle
  • Electrodes for administering the pulses (available from multiple sources, e.g., Harvard Apparatus has both plate and needle electrodes)
  • Electroporation power source
  • Additional reagents and equipment for harvesting tissue or evaluating expression levels and efficiency (Lucas and Heller, ; Heller et al., )

Alternate Protocol 1: Electroporation into Plant Protoplasts

  • 5‐mm strips (1 g dry weight) sterile plant material
  • Protoplast solution (see recipe)
  • Plant electroporation buffer (see recipe)
  • Rotary shaker
  • 80‐µm‐mesh nylon screen
  • 15‐ml conical centrifuge tube, sterile
  • Centrifuge with Beckman JS‐4.2 rotor
  • Additional reagents and equipment for counting cells with a hemacytometer (Elbing and Brent, ) and plant RNA preparation (Ausubel et al., )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Abdulhaqq, S.A. and Weiner, D.B. 2008. DNA vaccines: Developing new strategies to enhance immune responses. Immunol. Res. 42:219‐232.
   Andre, F. and Mir, L.M. 2004. DNA electrotransfer: Its principles and an updated review of its therapeutic applications. Gene Ther. 11:S33‐S42.
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. 2001. Phenol/SDS method for plant RNA preparation. Curr. Protoc. Mol. Biol. 9:4.3.1‐4.3.4.
   Bloch, K.D. and Grossmann, B. 2001. Digestion of DNA with restriction endonucleases. Curr. Protoc. Mol. Biol. 31:3.1.1‐3.1.21.
   Bodles‐Brakhop, A.M., Heller, R., and Draghia‐Akli, R. 2009. Electroporation for the delivery of DNA‐based vaccines and immunotherapeutics: Current clinical developments. Mol. Ther. 17:585‐592.
   Bronson, S.K. and Smithies, O. 1994. Altering mice by homologous recombination using embryonic stem cells. J. Biol. Chem. 269:27155‐27158.
   Chang, D.C. 1989. Cell poration and cell fusion using an oscillating electric field. Biophys. J. 56:641‐652.
   Chu, G., Hayakawa, H., and Berg, P. 1987. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 15:1311‐1326.
   Elbing, K. and Brent, R. 2002. Growth in liquid media. Curr. Protoc. Mol. Biol. 59:1.2.1‐1.2.2.
   Fromm, M., Taylor, L.P., and Walbot, V. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. U.S.A. 82:5824‐5828.
   Fuller, S.A., Takahashi, M., and Hurrell, J.G. 2001a. Preparation of myeloma cells. Curr. Protoc. Mol. Biol. 18:11.5.1‐11.5.3.
   Fuller, S.A., Takahashi, M. and Hurrell, J.G. 2001b. Cloning of hybridoma cell lines by limiting dilution. Curr. Protoc. Mol. Biol. 1:11.8.1‐11.8.2.
   Glasspool‐Malone, J., Somiari, S., Drabick, J., and Malone, R. 2000. Efficient nonviral cutaneous transfection. Mol. Ther. 2:140‐146.
   Gothelf, A., Mir, L.M., and Gehl, J. 2003. Electrochemotherapy: Results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat. Rev. 29:371‐387.
   Heilig, J., Elbing, K.L., and Brent, R. 2001. Large‐scale preparation of plasmid DNA. Curr. Protoc. Mol. Biol. 41:1.7.1‐1.7.16.
   Heller, L.C. and Heller, R. 2006. In vivo electroporation for gene therapy. Hum. Gene Ther. 17:890‐897.
   Heller, L.C., Jaroszeski, M.J., Coppola, D., McCrae, A.N., Hickey, J., and Heller, R. 2006. Optimization of cutaneous electrically mediated plasmid DNA delivery using a novel electrode. Gene Ther. 14:275‐280.
   Heller, R., Jaroszeski, M., Atkin, A., Moradpour, D., Gilbert, R., Wands, J., and Nicolau, C. 1996. In vivo gene electroinjection and expression in rat liver fed. FEBS Lett. 389:225‐228.
   Hirao, L.A., Wu, L., Khan, A.S., Satishchandran, A., Draghia‐Akli, R., and Weiner, D.B. 2008. Intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques. Vaccine 26:440‐448.
   Joyner, A.L. 2000. Gene Targeting. Oxford University Press, Oxford, United Kingdom.
   Kingston, R.E., Sheen, J., and Moore, D. 2001. Isotopic assays for reporter gene activity. Curr. Protoc. Mol. Biol. 63:9.7.1‐9.7.11.
   Lucas, M.L. and Heller, R. 2001. Immunomodulation by electrically enhanced delivery of a plasmid encoding IL‐12 to murine skeletal muscle. Mol. Ther. 3:47‐53.
   Mir, L.M., Bureau, M.F., Gehl, J., Rangara, R., Rouy, D., Caillaud, J‐M., Delaere, P., Branellec, D., Schwartz, B., and Scherman, D. 1999. High‐efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc. Natl. Acad. Sci. U.S.A. 96:4262‐4267.
   Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solutions. Curr. Protoc. Mol. Biol. 59:2.1.1‐2.1.10.
   Neumann, E., Schaefer‐Ridder, M., Wang, Y., and Hofschneider, P.H. 1982. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1:841‐845.
   Nishi, T., Yoshizato, K., Yamashiro, S., Takeshima, H., Sato, K., Hamada, K., Kitamura, I., Yoshimura, T., Saya, H., Kuratsu, J., and Ushio, Y. 1996. High‐efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer Res. 56:1050‐1055.
   Ou‐Lee, T.M., Turgeon, R., and Wu, R. 1986. Uptake and expression of a foreign gene linked to either a plant virus or Drosophila promoter in protoplasts of rice, wheat and sorghum. Proc. Natl. Acad. Sci. U.S.A. 83:6815‐6819.
   Potter, H. 1988. Electroporation in biology: Methods, applications, and instrumentation. Anal. Biochem. 174:361‐373.
   Potter, H., Weir, L., and Leder, P. 1984. Enhancer‐dependent expression of human κ immunoglobulin genes introduced into mouse pre‐B lymphocytes by electroporation. Proc. Natl. Acad. Sci. U.S.A. 81:7161‐7165.
   Rhodes, C.A., Pierce, D.A., Mettler, I.J., Mascarenhas, D., and Detmar, J.J. 1988. Genetically transformed maize plants from protoplasts. Science 240:204‐207.
   Roos, A.K., Moreno, S., Leder, C., Pavlenko, M., King, A., and Pisa, P. 2006. Enhancement of cellular immune response to a prostate cancer DNA vaccine by intradermal electroporation. Mol. Ther. 13:320‐327.
   Saunders, J.A., Matthews, B.F., and Miller, P.D. 1989. Plant gene transfer using electrofusion and electroporation. In Electroporation and Electrofusion in Cell Biology (E., Neumann, A.E., Sowers, and C.A., Jordan, eds.) pp. 343‐354. Plenum, New York.
   Sheen, J. 1990. Metabolic repression of transcription in higher plants. Plant Cell 2:1027‐1038.
   Titomirov, A.V., Sukharev, S., and Kistanova, E. 1991. In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim. Biophys. Acta 1088:131‐134.
   Trollet, C., Bloquel, C., Scherman, D., and Bigey, P. 2006. Electrotransfer into skeletal muscle for protein expression. Curr. Gene Ther. 6:561‐578.
   Wong, T.K. and Neumann, E. 1982. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107:584‐587.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library