Neuronal Transfection Using Particle‐Mediated Gene Transfer

Donald C. Lo1

1 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.15
DOI:  10.1002/0471142301.ns0315s05
Online Posting Date:  May, 2001
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Abstract

This unit describes the use of particle‐mediated gene transfer (also known as biolistics) for the transfection of neuronal cell lines and brain slices. Like nuclear microinjection of DNA, biolistics results in the direct introduction of DNA into the nucleus; it is perhaps for this reason that biolistics works as well in mitotic cells as in postmitotic cells such as skeletal muscle, skin, liver, and neurons. The basic principle of biolistics is to accelerate micron‐sized gold particles coated with DNA towards target cells or tissue. Cells penetrated by these particles have a high likelihood of being transfected by the DNA thus introduced. The motive force for particle acceleration can come from a variety of sources, the most widely used is described in this unit and is a supersonic shock wave generated by the rupture of a kapton membrane induced by high‐pressure helium. Another option included in this unit is to propel the gold particles by gas jet entrainment.

     
 
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Table of Contents

  • Basic Protocol 1: Gene Delivery Using a Helium‐Rupture Biolistics Device
  • Basic Protocol 2: Gene Delivery Using a Hand‐Held Gas‐Entrainment Biolistics Device
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Gene Delivery Using a Helium‐Rupture Biolistics Device

  Materials
  • 1.6‐µm gold particles (e.g., Bio‐Rad or Strem Chemicals)
  • Absolute ethanol
  • 0.5 to 1 µg/µl supercoiled plasmid DNA of interest
  • 2.5 M CaCl 2
  • 1 M spermidine (free base)
  • Isopropanol
  • Vacuum grease
  • Neuronal cells of interest (plated in individual tissue culture dishes up to 10 cm in diameter) or brain slices (in organotypic interface culture on 35‐mm transwell inserts)
  • Bath ultrasonicator
  • 10‐cm or larger plastic petri dish
  • 25‐mm kapton macrocarrier disks, commercially available (Bio‐Rad) or cut from 1‐mil (0.001‐in. or 0.025‐mm) kapton sheets (e.g., AIN Plastics)
  • Desiccated glove box (or equivalent dry environment) with vortex mixer
  • Helium‐rupture biolistics machine (Bio‐Rad PDS‐1000/He), with vacuum pump, helium tank and regulator, second low‐pressure helium tank (used for brain slices only), rupture disks and retaining cap, torque wrench, macrocarrier holders
  • 100‐µm‐opening nylon mesh (e.g., Small Parts; for brain slices only)
  • Stainless steel stopping screens (Bio‐Rad)
  • ∼2 × 2 × 0.3–cm blocks of 2% agarose (for brain slices only)
  • ∼2 × 2 × 1–cm aluminum block (for brain slices only)

Basic Protocol 2: Gene Delivery Using a Hand‐Held Gas‐Entrainment Biolistics Device

  Materials
  • Dry nitrogen
  • 1.6‐µm gold particles (e.g., Bio‐Rad or Strem Chemicals)
  • 0.05 M spermidine (free base, store aliquots at −70°C)
  • ≥1 µg/µl supercoiled plasmid DNA of interest
  • 1 M CaCl 2 in water
  • Absolute ethanol
  • 0.05 mg/ml PVP in absolute ethanol (see recipe)
  • Neuronal cells of interest (plated in individual or clusters of 3.5‐cm tissue culture dishes) or brain slices (in organotypic interface culture on 35‐mm transwell inserts)
  • Helios Gene Gun (Bio‐Rad) with battery, magazine, high‐pressure helium source, and regulator
  • Tefzel tubing and tubing preparation station (Bio‐Rad)
  • 10‐ml syringe with silicone adapter tube
  • Peristaltic pump set to draw liquid at 5.5 to 6.0 ml/min
  • Tubing cutter (Bio‐Rad)
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Figures

Videos

Literature Cited

Literature Cited
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