Ex Vivo Electroporation of Chick Embryo Cerebellar Slices: A Method to Introduce Plasmid DNA Encoding Green Fluorescent Protein to Visualize Granular Cell Development

Samenvatting

Source: Hanzel, M. et al. Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors. J. Vis. Exp. (2015)

In this video, we perform electroporation of chick embryo cerebellum slices with reporter gene plasmids. The technique facilitates visualization of the development and migration of granule cell precursors to form granular layer.

Protocol

1. Electroporation of Slices

1. Construct an electroporation chamber by fixing the anode of an electroporator to the base of a 60 mm Petri dish with insulation tape. Add approximately 1 mL of HBSS to cover the electrode.
2. Place a 0.4 µm culture insert on top of the electrode covered in HBSS. Allow the culture insert to rest on the electrode making sure there is always contact between the insert and the electrode.
   NOTE: In this setup, the culture insert with the slices will rest upon the surface of the solution, maintaining the circuit but allowing spatial targeting of the cathode.
3. Transfer identified slices (up to five per culture insert) onto culture insert using 3 mL Pasteur pipette with the cut tip. Separate and allow to settle onto culture insert in a sagittal orientation.
4. Using a pipette, remove excess HBSS so that slices are no longer bathed in solution.
5. Using a P10 pipette tip, pipette DNA (at a concentration of 1 µg/µL) diluted with 20% fast green over the surface of targeted region of a slice. 20% fast green ensures viscous DNA solution and prevents prohibitively wide dispersal of DNA. Add approximately 5 µL DNA/fast green solution to each slice (Figure 1B).
6. Place the cathode over desired targeted tissue and electroporate with 3 x 10 V, 10 msec duration pulses. Avoid direct contact of the cathode with the tissue. Instead, take advantage of surface tension of the liquid to maintain conductance (place the cathode as close to the tissue as possible without actually touching the tissue).
7. Repeat DNA delivery and electroporation to multiple regions of EGL on each individual cerebellar slice as desired.
8. Upon completion of electroporation, transfer culture insert to 30 mm Petri dish.
9. To each culture, add 1 mL of pre-warmed culture medium (Basal Medium Eagle, 0.5% (w/v) D-(+)-glucose, 1% B27 supplement, 2 mM L-Glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin) underneath the culture insert such that the culture insert is in contact with medium, but slices are not bathed in it.
10. Incubate cultures at 37 °C/6% CO₂ for up to 3 days. Replace all of the culture medium every 24 hr with fresh pre-warmed medium.
11. Following culture, fix slices on culture inserts for 1 hr in 4% paraformaldehyde (or O/N at 4 °C) in a fresh 30 mm dish with no culture media.

Resultaten

Figure 1. The electroporation chamber set up. (A) A picture of the custom-made electroporation chamber. The chamber consists of an anode of an electroporator placed securely on the base of a 60 mm Petri dish. The dish cont...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Basal Medium Eagle (Gibco)	 Life Technologies	 41010-026
L-glutamine	 Sigma	 G7513
Penicillin/Streptomycin	 Sigma	 P4333
0.4 μm culture insert	 Millipore	 PICM0RG50
TSS20 Ovodyne electroporator	 Intracel	 01-916-02	Use 3 x 10 V 10 msec pulses for electroporation.