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Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.
Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell’s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.
Zebrafish are excellent animal models to examine brain regeneration after a stab wound injury. In comparison to mammals, on the evolutionary ladder, less evolved species such as zebrafish generally show higher rates of constitutive neurogenesis and broader areas of adult neural stem cell residence, leading to constant generation of new neurons throughout most brain areas in the adult life. This feature appears to correlate with significantly higher regenerative capacity of zebrafish in comparison to mammals1, as zebrafish have remarkable potential to efficiently generate new neurons in most brain injury models studied2
All animals used in this protocol were kept under standard husbandry conditions, and experiments have been performed according to the handling guidelines and regulations of EU and the Government of Upper Bavaria (AZ 55.2-1-54-2532-0916).
1. Preparation of Plasmid Mixture for Electroporation
The described electroporation method allows delivery of plasmid DNA into ependymoglial cells, which are located superficially in the zebrafish telencephalon and just under the dorsal ependymal cell layer (Figure 1A).
If the result of electroporation is positive, labeled single ependymoglial cells (red cells in Figure 2A,B) can be observed among other ependymoglial cells (white in
This electroporation protocol is a reliable in vivo method of labelling individual ependymoglial cells. The protocol may need a further adaptation to label other cell types such as neurons or oligodendrocytes. To achieve successful labelling, plasmids containing different promoters can be used. Chicken-beta actin promoter, eF1alpha, CMV and ubiquitin promoter have been previously used to drive the expression of different transgenes in ependymoglia and their progeny23. However, different kinetics o.......
Special thanks to James Copti for editing of the manuscript. We also gratefully acknowledge funding to JN from the German Research foundation (DFG) by the SFB 870 and SPP “Integrative Analysis of Olfaction” and SPP 1738 “Emerging roles of non-coding RNAs in nervous system development, plasticity & disease”, SPP1757 “Glial heterogeneity”, and Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy – ID 390857198).
....Name | Company | Catalog Number | Comments |
Reagent/Material | |||
Fast Green | Sigma-Aldrich | F7258-25G | For coloring plasmid solution |
MS222 | Sigma-Aldrich | A5040-25G | MS222 should be stored at RT (up to two weeks) and protected from light |
Ultrasound gel | SignaGel, Parker laboratories INC. | 15-60 | Electrode Gel |
Equipment | |||
Air pump | TetraTec APS 50, 10l-60l | Can be bought in the pet shops | |
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 | |
Injection device | FemtoJet 4i, Eppendorf | 5252000013 | |
Standard Wall Borosillicate Glass Capillary | Warner Instruments | 64-0766 | Model No: G100-4 |
Microloader tips | Eppendorf | 5242956003 | |
Micro-knife | Fine Science Tools | 10056-12 | |
Joystick micromanipulator | Narishige Japan | MN - 151 | |
Needle holder | FemtoJet 4i, Eppendorf | 5252000013 | Needle holder comes together with the injection device |
Needle pulling device | Narishige Japan | Model No: PC-10 | The PC-10 was discontinued by Narishige in 2017 and replaced by the PC-100 |
Petri dishes | Greiner Bio-One International | 633161 |
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