1. Electroporation
<ol>
	<li>Remove the fish from the injection set-up while still holding it in the sponge.</li>
	<li>Immerse the inner side of the tip of the electrodes in the ultrasound gel.</li>
	<li>Cover the fish telencephalon with a small amount of ultrasound gel.</li>
	<li>Position the fish head between the electrodes, placing the positive electrode at the ventral side of the fish&#8217;s head and the negative electrode on the dorsal side (Figure 1C), while still holding the fish&#8217;s body in the sponge. This sets the direction of the flow of the current necessary to electroporate ependymoglia positioned at subventricular zone.</li>
	<li>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.</li>
</ol>

<table><tbody><tr><td>Ultrasound gel</td><td>SignaGel, Parker laboratories INC</td><td>15-60</td><td>Electrode Gel</td></tr><tr><td>BTX Tweezertrodes Electrodes</td><td>Platinum Tweezertrode, BTX Harvard Apparatus</td><td>45-0486</td><td>1mm diameter</td></tr><tr><td>Electroporation device</td><td>BTX ECM830 Square Wave Electroporation System, BTX Harvard Apparatus</td><td>45-0662</td><td></td></tr></tbody></table>

in vivo electroporation: a site-specific method to transfect zebrafish

Source: Durovic, T., et al. <a href="https://www.jove.com/v/60066/" target="_blank">Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon</a>. J. Vis. Exp. (2019).
This video describes in vivo electroporation, which is a technique for DNA delivery into ependymoglial cells in adult zebrafish.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20193/20193fig1.jpg" /> 
Figure 1: Schematic representation of coronal section of the everted zebrafish telencephalon.&#160;(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...

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

Source: Durovic, T., et al. Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon. J. Vis. Exp. (2019).
This ...

- First, place an anesthetized fish with the dorsal side facing up into a pre-wetted sponge. Before electroporation, inject genetic material into the target tissue, in this case, a part of the forebrain called the telencephalon. Next, put a small amount of ultrasound gel on the head. Also, apply the gel to the tip of the electrodes to facilitate efficient operation. Place the positive electrode on the ventral side of the head and the negative electrode on the dorsal side.
Now, press the electrodes gently against the head and administer several pulses of electric current. The current creates temporary pores in the plasma membrane of electroporated cells and makes them permeable. The current also facilitates the movement of negatively charged particles, such as DNA expression plasmids or RNAi oligonucleotides present in the surrounding, into the cells. In the following example, we will use electroporation to introduce a recombinant plasmid into ependymoglial cells, stem cells responsible for the generation of new neurons.
- Cover the fish telencephalon with a small amount of ultrasound gel. For electroporation of the plasmid-injected animal, remove the sponge from the ejection set up and immerse the inner side of the electrode tips into ultrasound gel. Position the fish head between the electrodes, with the positive electrode at the ventral side of the head and the negative electrode on the dorsal side. Then, while still holding the fish&#39;s body in the sponge, press the electrodes gently and precisely against the telencephalon and administer the current with the foot pedal, holding the electrodes in place until all five pulses are finished.

in-vivo-electroporation-a-site-specific-method-to-transfect-zebrafish

Research

JoVE Encyclopedia of Experiments

Biology

Danio rerio (zebrafish)

Adult

2.4K 次观看. - 首先，将一条背侧朝上的麻醉鱼放入预先润湿的海绵中。在电穿孔之前，将遗传物质注射到目标组织中，在这种情况下，是前脑的一部分，称为端脑。接下来，将少量超声凝胶涂在头部。此外，将凝胶涂抹在电极尖端以促进高效作。将正极放在头部的腹侧，将负极放在背侧。 现在，将电极轻轻按压在头部并施加几次电流脉冲。电流在电穿孔细胞的质膜上产生临时孔，?...

体内研究电穿孔:一种转染斑马鱼的位点特异性方法

This video describes in vivo electroporation, which is a technique for DNA delivery into ependymoglial cells in adult zebrafish.

Overview

Protocol

<img title="figure-results-25" alt="figure-results-25" class="xfigimg" src="/files/ftp_upload/20193/20193fig1.jpg" /> 
Figure 1: Schematic representation of coronal section of the everted zebrafish telencephalon.&#160;(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...

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

本文内容

Overview

研究方案

结果

材料

参考文献

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