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

Protokol

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.

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Sonuçlar

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 contains approximately 1 mL of HBSS to cover the electrode. The culture insert should rest on the electrode with constant contact between the insert and the electrode, maintaining the circuit but allowing spatial targeting of the cathode, which is manipulated by hand. (B) A picture of slices being electroporated. Slices are covered with the DNA/fast green solution. The slices are electroporated as desired: electroporation can be targeted to one folium or multiple locations. After electroporation the insert is placed in a 30 mm Petri dish with pre-warmed culture medium and cultured in the incubator. 1. The anode 2. The cathode 3. Culture insert 4. Petri dish with 1 mL HBSS 5. Dissecting microscope 6. Individual slices from tissue chopper 7. DNA solution with fast green dye.

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Malzemeler

Name	Company	Catalog Number	Comments
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.