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Neuronal Transfection Using Particle‐Mediated Gene Transfer

Donald C. Lo¹

¹Duke 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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Unit Introduction
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 CaCl₂
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)
³1 µg/µl supercoiled plasmid DNA of interest
1 M CaCl₂ 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

Figure 3.15.1

Basic procedure of particle-mediated gene transfer in brain slices or cultured cells. Gold particles (1.6-µm diameter) are coated with plasmid DNA by precipitation. The DNA-coated particles are loaded onto a kapton membrane (macrocarrier) in an ethanol suspension and allowed to dry. The macrocarrier is then mounted in the biolistics device between the rupture disk and the stopping screen. Pressure on the rupture disk is increased using compressed helium until it ruptures; the resulting pressure shock wave propels the macrocarrier against the stopping screen at high velocity. The stopping screen allows only the particles to pass through to bombard brain slices or cultured cells placed in their trajectory. Bombarded cells or tissues are assayed for transfection 16 to 72 hr later by visualization of reporters such as -galactosidase.

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Figure 3.15.2

Low-power view of a brain slice taken from P14 ferret visual cortex transfected with a lacZ expression construct and processed by Xgal histochemistry 20 hr posttransfection. Approximately 3000 neurons were transfected in this slice.

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Figure 3.15.3

At higher power, the morphological details of transfected neurons in a brain slice becomes evident. Note the gold particles scattered throughout the field, appearing under these optical conditions as small black dots. Reprinted with permission from McAllister et al. (1997).

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Figure 3.15.4

Live PC12 cells cotransfected three days prior with DNA constructs encoding the TrkC receptor and GFP. Note that only the GFP-expressing cells seen under green fluorescence (A) have extended neuronal processes in response to NT-3 (B). Reprinted with permission from Sherwood et al. (1997).

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Literature Cited

Literature Cited
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	McAllister, A.K., Katz, L.C., and Lo, D.C. 1996. Neurotrophin regulation of cortical dendritic growth requires activity. Neuron 17:1057-1064.
	McAllister, A.K., Katz, L.C., and Lo, D.C. 1997. Opposing roles for endogenous BDNF and NT-3 in regulating cortical dentritic growth. Neuron 18:767-778.
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	Sherwood, N.T., Lesser, S.S., and Lo, D.C. 1997. Neurotrophin regulation of ionic currents and cell size depends on cell context. Proc. Natl. Acad. Sci. U.S.A. 94:5917-5922.
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