Transfection by Electroporation

Huntington Potter1, Richard Heller2

1 Rocky Mountain Alzheimer's Disease Center, Anschutz Medical Campus, University of Colorado, Aurora, Colorado, 2 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.3
DOI:  10.1002/cpmb.48
Online Posting Date:  January, 2018
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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. © 2018 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
  • Alternate Protocol 1: Preparation of Cells for Electroporation in Tissue Culture Plates
  • Support Protocol 1: Determination of Correct Electroporation Parameters
  • Basic Protocol 2: Electroporation into Muscle or Skin
  • Alternate Protocol 2: 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 3F; Phelan and May, ) without and with appropriate selective agents (unit 9.5; Mortensen and Kingston, )
  • Electroporation buffer (see recipe), ice‐cold
  • Linear or supercoiled, purified DNA preparation (see step 7)
  • Centrifuge with Beckman JS‐4.2 rotor or equivalent (not necessary for adherent cells that will not be transfected in a cuvette)
  • Electroporation cuvettes (Bio‐Rad, cat. no. 165‐2088), tissue culture plates, or flow‐through system (BTX, cat. no. 45‐0130, 45‐0450, 45‐0466; Nucleofector Y Unit from Lonza; Maxcyte STX, VLX, GT systems), electroporation plates [BTX, cat. no. 45‐0466 (25‐well) or 45‐0450 (96‐well], 96‐well shuttle system (Lonza), conductive polymer dipping electrode arrays for 24‐well plates (Lonza), petri pulser electrode array (BTX, cat no. 45‐0130 or 45‐0100), adherent cell electrodes (BTX, cat no. 45‐0530)
  • Electroporator (Bio‐Rad Gene Pulser X‐Cell, BTX 830 or 630, Lonza Nucleofector,or equivalent) and power source
  • Additional reagents and equipment for stable transformation in selective medium (unit 9.5; Mortensen and Kingston, ) and for harvesting transfected cells [unit 9.6 (Kain and Ganguly, ), unit 9.7 (Kingston et al., ), unit 9.7 (Brasier and Fortin, ), unit 9.8 (Kingston, ), and unit 14.6 (Goldstein and Watkins, )]

Alternate Protocol 1: Preparation of Cells for Electroporation in Tissue Culture Plates

  Additional Materials (also see protocol 1)
  • Appropriate tissue culture plates

Support Protocol 1: Determination of Correct Electroporation Parameters

  • 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‐cc 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, L.C., et al., )

Basic Protocol 2: Electroporation into Muscle or Skin

  • 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
  • Sterile 15‐ml conical centrifuge tube
  • Centrifuge with Beckman JS‐4.2 rotor
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.2; Elbing and Brent, ) and plant RNA preparation (unit 4.3; Anonymous, )
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Literature Cited

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