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
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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

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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
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Basic Protocol 1: Electroporation into Mammalian Cells

  • 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

  • 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., )
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Literature Cited

Literature Cited
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