In Vivo Electroporation: A Site-Specific Method to Transfect Zebrafish

Protokół

1. Electroporation

1. Remove the fish from the injection set-up while still holding it in the sponge.
2. Immerse the inner side of the tip of the electrodes in the ultrasound gel.
3. Cover the fish telencephalon with a small amount of ultrasound gel.
4. Position the fish head between the electrodes, placing the positive electrode at the ventral side of the fish’s head and the negative electrode on the dorsal side (Figure 1C), while still holding the fish’s body in the sponge. This sets the direction of the flow of the current necessary to electroporate ependymoglia positioned at subventricular zone.
5. Press the electrodes gently and precisely against the telencephalon (Figure 1 C). Administer the current with the foot pedal. Hold the electrodes in place until all five pulses are finished.

Wyniki

Figure 1: Schematic representation of coronal section of the everted zebrafish telencephalon. (A) Scheme of a coronal section of zebrafish telencephalon, highlighting the position of ependymoglial cells, which are lining ventricular surface and building the ventral ventricular wall. Dorsal ependymal layer is bridging the two hemispheres and covering the ventricle (V), located in between two cell...

Materiały

Name	Company	Catalog Number	Comments
Ultrasound gel	SignaGel, Parker laboratories INC	15-60	Electrode Gel
BTX Tweezertrodes Electrodes	Platinum Tweezertrode, BTX Harvard Apparatus	45-0486	1mm diameter
Electroporation device	BTX ECM830 Square Wave Electroporation System, BTX Harvard Apparatus	45-0662