ex vivo electroporation of chick embryo cerebellar slices: a method to introduce plasmid dna encoding green fluorescent protein to visualize granular cell development

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

Construct an electroporation chamber by fixing the anode of an electroporator to the base of a 60-millimeter Petri dish using insulation tape. Add approximately 1 milliliter of HBSS to cover the electrode. Next, place a 0.4-micrometer culture insert on top of the electrode covered in HBSS.
Use a 3-milliliter Pasteur pipette with a cut tip to transfer the identified slices, up to 5 per insert onto the culture insert. Then, separate the slices and allow them to settle onto the culture insert in a sagittal orientation. Using a pipette, remove excess medium from the brain slices. Allow the insert to rest on the electrode so that there is contact between the insert and the electrode.
In this setup, the culture insert with the slices will rest upon the surface of the medium, maintaining the circuit but allowing spatial targeting of the cathode. Using a P10 pipette tip, pipette 5 microliters of DNA diluted with 20% Fast Green over the surface of a targeted region of each slice. The addition of Fast Green ensures that the DNA solution is viscous enough to prohibit wide dispersal of the DNA.
Then, place the cathode over the desired targeted tissue and electroporate the samples. Avoid direct contact of the cathode with the tissue by placing the cathode as close to the tissue as possible without actually touching it. Repeat the electroporation in multiple regions of the external granular layer on each individual cerebellar slice as desired.
When finished, transfer the culture insert to a 30-millimeter Petri dish. To each culture, add 1 milliliter of pre-warmed culture medium underneath the culture insert. Make sure that the insert does not float on the medium. The culture insert should be in contact with the medium, but the slices should not be bathed in it.

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1. Electroporation of Slices
<ol>
	<li>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.</li>
	<li>Place a 0.4 &#181;m 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.</li>
	<li>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.</li>
	<li>Using a pipette, remove excess HBSS so that slices are no longer bathed in solution.</li>
	<li>Using a P10 pipette tip, pipette DNA (at a concentration of 1 &#181;g/&#181;L) 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 &#181;L DNA/fast green solution to each slice (Figure 1B).</li>
	<li>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).</li>
	<li>Repeat DNA delivery and electroporation to multiple regions of EGL on each individual cerebellar slice as desired.</li>
	<li>Upon completion of electroporation, transfer culture insert to 30 mm Petri dish.</li>
	<li>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 &#956;g/mL streptomycin) underneath the culture insert such that the culture insert is in contact with medium, but slices are not bathed in it.</li>
	<li>Incubate cultures at 37 &#176;C/6% CO2&#160;for up to 3 days. Replace all of the culture medium every 24 hr with fresh pre-warmed medium.</li>
	<li>Following culture, fix slices on culture inserts for 1 hr in 4% paraformaldehyde (or O/N at 4 &#176;C) in a fresh 30 mm dish with no culture media.</li>
</ol>

<table><tbody><tr><td>Basal Medium Eagle (Gibco)</td><td>&amp;nbsp;Life Technologies</td><td>&amp;nbsp;41010-026</td><td></td></tr><tr><td>L-glutamine</td><td>&amp;nbsp;Sigma</td><td>&amp;nbsp;G7513</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>&amp;nbsp;Sigma</td><td>&amp;nbsp;P4333</td><td></td></tr><tr><td>0.4 &amp;mu;m culture insert</td><td>&amp;nbsp;Millipore</td><td>&amp;nbsp;PICM0RG50</td><td></td></tr><tr><td>TSS20 Ovodyne electroporator</td><td>&amp;nbsp;Intracel</td><td>&amp;nbsp;01-916-02</td><td>Use 3 x 10 V 10 msec pulses for electroporation.</td></tr></tbody></table>

Source: Hanzel, M. et al.&#160;<a href="https://www.jove.com/video/53421" target="_blank">Ex Vivo&#160;Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.</a>&#160;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.

<img alt="Figure 1" src="/files/ftp_upload/20695/20695_Figure1.jpg" /> 
Figure 1.&#160;The electroporation chamber set up.&#160;(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...

Source: Hanzel, M. et al.&#160;Ex Vivo&#160;Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.&#160;J. ...

Cerebellar granule cells are the brain&#39;s most abundant type of neurons. The external granular layer, EGL, is composed solely of granular cell precursors, GCPs, that differentiate and migrate inwardly to form the inner granular layer, IGL.
To visualize granular cell development, prep an electroporation chamber by attaching an anode to the base of a Petri dish. Add culture medium to cover the electrode. Now, place a culture insert on the medium-covered electrode. Transfer the chick embryo cerebellum slices onto the culture insert in a sagittal orientation.
The sagittal plane allows the target layer-EGL to be identified against different cerebellum layers. Add a viscous dye solution containing a GFP plasmid DNA over EGL. The GFP plasmid DNA encodes for a green fluorescent protein that distributes homogeneously within the cell cytoplasm and enables live-cell imaging.&#160;
Next, place a cathode in the medium near EGL but not directly on the tissue. Then, apply multiple electric pulses to different EGL regions. The electric pulses induce temporary pores in the cell membrane, allowing cells to take up the plasmid DNA. Subsequently, plasmid DNA enables GFP expression in granular cells.
Image the cerebellar slices under a fluorescence microscope to observe granular cell differentiation and migration pattern across different stages of cell development.

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

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

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