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Summary

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.

Abstract

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.

Introduction

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

Protocol

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

  1. Dilute the plasmid of interest in sterile water and add fast green stain stock solution [1 mg/mL]. Make sure that the final concentration of the plasmid is  ∼1 µg/µL. Add th.......

Representative Results

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

Discussion

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.......

Acknowledgements

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).

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Materials

NameCompanyCatalog NumberComments
Reagent/Material
Fast GreenSigma-AldrichF7258-25GFor coloring plasmid solution
MS222Sigma-AldrichA5040-25GMS222 should be stored at RT (up to two weeks) and protected from light
Ultrasound gelSignaGel, Parker laboratories INC.15-60Electrode Gel
Equipment
Air pumpTetraTec APS 50, 10l-60lCan be bought in the pet shops
BTX Tweezertrodes ElectrodesPlatinum Tweezertrode, BTX Harvard Apparatus45-04861mm diameter
Electroporation deviceBTX ECM830 Square Wave Electroporation System, BTX Harvard Apparatus45-0662
Injection deviceFemtoJet 4i, Eppendorf5252000013
Standard Wall Borosillicate Glass CapillaryWarner Instruments64-0766Model No: G100-4
Microloader tipsEppendorf5242956003
Micro-knifeFine Science Tools10056-12
Joystick micromanipulatorNarishige JapanMN - 151
Needle holderFemtoJet 4i, Eppendorf5252000013Needle holder comes together with the injection device
Needle pulling deviceNarishige JapanModel No: PC-10The PC-10 was discontinued by Narishige in 2017 and replaced by the PC-100
Petri dishesGreiner Bio-One International633161

References

  1. Kaslin, J., Ganz, J., Brand, M. Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain. Philosophical Transactions of the Royal Society of London Series B Biological Sciences. 363 (1489), 101-122 (2008).
  2. Baumgart, E. V., Barbosa, J. S., Bally-Cuif, L., Gotz, M., Ninkovic, J.

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