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 &#8220;Integrative Analysis of Olfaction&#8221; and SPP 1738 &#8220;Emerging roles of non-coding RNAs in nervous system development, plasticity &#38; disease&#8221;, SPP1757 &#8220;Glial heterogeneity&#8221;, and Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy &#8211; ID 390857198).

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
<ol>
	<li>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&#160; &#8764;1 &#181;g/&#181;L. Add the stain at a concentration of no more than 3%, as its only purpose is to color the solution and visualize ventricular injection.</li>
	<li>Once prepared, mix the plasmid solution by pipetting up and down several times or by finger tapping. Store at room temperature (RT) until usage. 
	NOTE: For co-electroporation of two plasmids into the same cell, ensure that the concentration of each individual plasmid used in the mixture is at least 0.8 ng/&#181;L with a molar ratio of 1:1 to obtain 80%&#8211;90% co-electroporation efficiency.</li>
</ol>
2. Preparations for Injection and Electroporation Procedure
<ol>
	<li>Prepare the glass capillaries (outer diameter 1 mm, inner diameter 0.58 mm) necessary for the injection in the needle pulling apparatus.</li>
	<li>In order to inject the correct amount of plasmid (see above), pull the capillary at a temperature of 68.5 &#176;C with two light and two heavy weights (see Table of Materials for puller specifications). 
	NOTE: In case a different puller is used, calibrate the capillary to deliver the appropriate volume of electroporation mix.</li>
	<li>Manually set the injection device to an injection pressure of 200 hPa (by turning the Pi knob) and constant pressure of 0 hPa. Manually set the injection time to manual mode and control the pressure with foot pedal.</li>
	<li>Set the electroporation device to &#8220;LV mode&#8221; with five pulses at 54&#8211;57 V (25 ms each with 1 s intervals). Connect the electrodes to the device.</li>
	<li>Prepare one fish tank with clean fish water, where the fish will be awakened from anesthesia after the electroporation procedure. Aerate the water by keeping the air stone attached to the air pump for the entire recovery period until the fish is fully awakened.</li>
	<li>Take a regular kitchen sponge and make a longitudinal cut in the sponge to hold fish into during the injection and electroporation procedure (see previous publication3). 
	NOTE: The kitchen sponge should be regularly washed or exchanged in order to remove potential toxic chemicals.</li>
	<li>Place a small amount of highly conductive multi-purpose ultrasound gel next to the injection and electroporation setup. 
	NOTE: This will ensure adequate electrical conductivity, and consequently, distribution of electroporated cells throughout the entire telencephalon.</li>
</ol>
3. Zebrafish Anesthesia
<ol>
	<li>Prior to anesthetization, prepare a stock solution of anesthesia with 0.2% MS222 in distilled water and adjust the pH to 7 with Tris-HCl buffer. Dilute this stock 1:10 (i.e., to 0.02% MS222) using fish water.</li>
	<li>Anesthetize the fish by keeping them in this working solution until the movement of the body and gills subsides (typically for a couple of minutes).</li>
</ol>
4. Plasmid Solution Injection
<ol>
	<li>Fill the prepared glass capillary with 10 &#181;L of plasmid solution using microloader tips. Avoid the formation of air bubbles inside the capillary.</li>
	<li>Press Menu/Change Capillary on the injection device. Insert and secure the needle into the needle holder.</li>
	<li>Under a stereomicroscope with a magnification of 3.2x or 4x, cut only the tip of the capillary using fine-end forceps. Switch the injection device from Change Capillary mode into Inject mode, then apply pressure with a foot pedal to ensure that the plasmid solution is running easily out of the needle and without hindrance.</li>
	<li>Transfer the fish from the husbandry tank to the container (plastic box) with anesthetic solution. Wait for a few minutes until movement of the gills subsides.</li>
	<li>Place fish into the pre-wetted sponge with the dorsal side facing up. Perform all the following injection steps under the stereomicroscope to ensure the accuracy of procedure.</li>
	<li>Using a dissecting micro-knife from stainless steel with 40 mm cutting edge and 0.5 mm thickness, create a small hole in the fish skull at the posterior side of the telencephalon (Figure 1B), just next to the border with optic tectum. 
	NOTE: This step should be performed carefully since the hole should be very small and superficial, penetrating solely the skull, to avoid brain damage.</li>
	<li>Tilt the fish as necessary and orient the tip of the glass capillary towards the skull in the correct angle to facilitate penetration of the capillary tip through the hole.</li>
	<li>Insert the tip of the capillary through the hole in the skull carefully until it reaches the telencephalic ventricle (see Figure 1B). This will require penetration through the dorsal ependymal cell layer. Be especially careful not to insert the capillary too deeply to avoid contact with the brain tissue. Keep the capillary precisely in between the hemispheres, remaining inside the ventricle just after piercing the dorsal ependymal layer. 
	NOTE: This is a very delicate step. Accuracy of this procedure is improved using pigmentation mutant lines such as brassy24, allowing better visualization of glass capillary position during injection.</li>
	<li>With the capillary tip inside of the ventricle, inject the plasmid solution by applying pressure with the foot pedal for about 10 s, which corresponds to approximately 1 &#181;L of plasmid solution. 
	NOTE: If changing the needle puller, capillaries or injector, the system should be calibrated in order to always deliver 1 &#181;L of plasmid solution. Calibration can be performed by measuring the diameter of the plasmid droplet expelled into a mineral oil (e.g., paraffin oil) and subsequently calculating the volume of the droplet. After 10 s of injection, there should be ~1 &#181;L of plasmid liquid expelled into the mineral oil.</li>
	<li>Confirm success of the previous step by observing the spread of liquid throughout the ventricle.</li>
</ol>
5. 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 1C). Administer the current with the foot pedal. Hold the electrodes in place until all five pulses are finished.</li>
</ol>
6. Fish Recovery
<ol>
	<li>Let the fish recover in the previously prepared, continuously aerated tank until it wakes up. Lidocaine gel could be applied on the skull in order to relieve any possibly developed pain.</li>
</ol>

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Authors have nothing to disclose.

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...

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 studied2,3,4,5,6,7,8. Here, the zebrafish telencephalon is studied, since it is a brain area with prominent neurogenesis in adulthood. These zones of adult neurogenesis are homologous to neurogenic zones in the adult mammalian brain9,10,11.
Abundant neurogenic areas in the zebrafish telencephalon are present due to the existence of radial glia like cells or ependymoglia cells. Ependymoglial cells act as resident adult neural stem cells and are responsible for generation of new neurons in both the intact and regenerating brain3,5. Lineage tracing experiments have shown that ventricular ependymoglia react to injury, then proliferate and generate new neuroblasts that migrate to the lesion site5. Due to the everted nature of zebrafish telencephalon, ependymoglial cells line the ventricular surface and build the ventral ventricular wall. The dorsal ventricular wall is formed by a dorsal ependymal cell layer (Figure 1A). Importantly, zebrafish ependymoglia embody the characteristics of both mammalian radial glia and ependymal cells. Long radial processes are a typical feature of radial glia cells, whereas cellular extensions and tight junctions (as well as their ventricular positions) are typical features of ependymal cells12,13,14. Therefore, these cells are referred to as ependymoglial cells.
To follow in vivo behavior of single ependymoglial cells during regeneration, they need to be reliably labeled. Various methods of in vivo cell labeling for fluorescent microscopy have been previously described, such as endogenous reporters or lipophilic dyes15. These methods, in contrast to electroporation, may require longer periods of time and often do not offer the possibility of single cell labeling or permanent long-term tracing. Electroporation, however (besides single cell labeling), offers the possibility of introducing new DNA into the host cell. Moreover, compared to other methods of DNA transfer into the cells, electroporation has been demonstrated to be one of the most efficient methods16,17,18,19.
Presented here is an electroporation protocol that has been refined for the purpose of labeling single ependymoglial cells in the adult zebrafish telencephalon. This protocol allows for the labelling of single ependymoglial cells in order to follow them over a long-term period20 or to manipulate specific pathways in a cell-autonomous manner21,22.

<table><tbody><tr><td>&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258-25G</td><td>For coloring plasmid solution</td></tr><tr><td>MS222</td><td>Sigma-Aldrich</td><td>A5040-25G</td><td>MS222 should be stored at RT (up to two weeks) and protected from light</td></tr><tr><td>Ultrasound gel</td><td>SignaGel, Parker laboratories INC.</td><td>15-60</td><td>Electrode Gel</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Air pump</td><td>TetraTec APS 50, 10l-60l</td><td></td><td>Can be bought in the pet shops</td></tr><tr><td>BTX Tweezertrodes Electrodes</td><td>Platinum Tweezertrode, BTX Harvard Apparatus</td><td>45-0486</td><td>1 mm 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><tr><td>Injection device</td><td>FemtoJet 4i, Eppendorf</td><td>5252000013</td><td></td></tr><tr><td>Standard Wall Borosillicate Glass Capillary</td><td>Warner Instruments</td><td>64-0766</td><td>Model No: G100-4</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Micro-knife</td><td>Fine Science Tools</td><td>10056-12</td><td></td></tr><tr><td>Joystick micromanipulator</td><td>Narishige Japan</td><td>MN - 151</td><td></td></tr><tr><td>Needle holder</td><td>FemtoJet 4i, Eppendorf</td><td>5252000013</td><td>Needle holder comes together with the injection device</td></tr><tr><td>Needle pulling device</td><td>Narishige Japan</td><td>Model No: PC-10</td><td>The PC-10 was discontinued by Narishige in 2017 and replaced by the PC-100</td></tr><tr><td>Petri dishes</td><td>Greiner Bio-One International</td><td>633161</td><td></td></tr></tbody></table>

electroporation method for in vivo delivery of plasmid dna in the adult zebrafish telencephalon

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&#8217;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.

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 <s...

Watch this Scientific Journal Video about Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon at JoVE.com

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

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 ...

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9.7K Views.  Helmholtz Zentrum München. 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.

Video: Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuroscience

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Immunology and Infection

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Biology

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Optimized Intraperitoneal Injection for Compound Delivery in Adult Zebrafish

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both&#160;methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

Medicine

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.

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

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Summary

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Chapters in this video

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

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