This work was supported by grants from the Deafness Research Foundation to Y-c S and the NSF (IOS 0930096) to KFB. KFB: NIH/NINDCD 2 RO1 DC04184, <a href="https://commons.era.nih.gov/commons/genericStatus.do?actionRole=nonPI&amp;applID=7854446&amp;uhf-token=Ugf7JvudZTCbTUW9jSAR1snMUgg%3D">3R01DC004184-08W1</a>, NSF DBI 0832862, NSF IOS 0930096

1. Making Electrodes for Electroporation
<ol>
<li>Cut 75 &mu;m diameter tungsten wire (A-M Systems, Inc. Carlsborg, WA) of suitable length (about 3-5 cm)</li>
<li>Put tungsten wire through molex cable connector (pin stamped brass)</li>
<li>Wrap tungsten wires and molex cable connector with shrink tubing (SPC Technology, Chicago, IL) and apply heat with Bunsen Burner or heat gun to seal the shrinking tubing to the wire</li>
<li>To sharpen the tip of tungsten wire, dip the wire into 1.0 N Sodium Hydroxide (Conrad et al. 1993) and, using a paper-clip as the other electrode, electrolyze the wire with a Square Wave Electroporator. The conditions we use are 5 50-Volt pulses each lasting 100 ms with 50 ms in between pulses. Electrodes should be sharpened to a diameter of 15-20 &mu;m</li>
<li>Keep the wires in a tray within grooves pressed into modelling clay prior to use in electroporation</li>
</ol>
2. Set Up Electroporation Station
<ol>
<li>Tape sharpened 75&mu;m tungsten electrodes to micromanipulators (World Precision instruments).</li>
<li>Place micromanipulators on either side of the dissecting microscope stage (Leica).</li>
<li>Connect the electrodes to a Protect CUY-21 Edit Square Wave Electroporator (Figure 1).</li>
<li>Turn on the electroporator and set up Parameters for electroporation, including voltage, pulse duration, and number of pulses. The most effective parameters for this experiment were found to be three 13-volt pulses which each lasted for 5.0ms, with 100ms in between each pulse. If electroporation creates air bubbles in the embryo, reduce the pulse length.</li>
</ol>
3. Microinjection of Morpholinos Into Zebrafish Otic Vesicle
<ol>
<li>Harvest zebrafish embryos from a breeding tank. Incubate the embryos in embryo-raising medium with 0.3 PPM methylene blue (Westerfield, 2005) at 28.5&deg;C overnight.</li>
<li>Pull glass needles with a Sutter P-97 electrode puller</li>
<li>Warm up an agarose gel base (1% agarose in fish water in a 10 mm Petri dish) to 37&deg;C in a water bath</li>
<li>Heat 1% low melting point agarose (LMPA) in fish water until melted and keep it warm in the 37&deg;C water bath.</li>
<li>Fill a glass needle with a morpholino solution (GeneTools, Corvallis, OR) and cut the tip to appropriate diameter. Mount the injection needle onto a micromanipulator (Kuhn).</li>
<li>Cut needle tip to approximately 10 &mu;m and inject into mineral oil to ensure that the tip allows sufficient delivery of morpholino solution.</li>
<li>Dechorinate embryos at 24 hours postfertilization (hpf) and anesthetize them with MS222 (tricaine, Sigma) in 0.3 PPM methylene blue fish water.</li>
<li>Align 3 to 5 anesthetized embryos onto the agarose gel base with the right side up for convenient uniform analysis</li>
<li>Put 1 to 2 drops of 1% LMPA over each embryo and let solidify to fix the embryo in place</li>
<li>Rotate the Petri dish so that the otic vesicle is on the right side</li>
<li>Place the needle into the lumen of the otic vesicle and inject the morpholino solution with a PV820 Pneumatic PicoPump (World Precision Instruments) with an attached nitrogen tank (Figure 2)</li>
</ol>
4. Electroporation Procedure
<ol>
<li>Move the mounted embryos to the electroporation station immediately after injection</li>
<li>Place the positive electrode onto the tissue just posterior to the otic vesicle, but do not penetrate; insert the negative electrode into the brain just anterior to the otic vesicle</li>
<li>Apply current using a foot pedal or by pushing a switch.</li>
<li>Pour fish water on agarose and remove embryos from agarose with swirling and a glass pipette. Use caution not to damage the embryos. For embryos that are difficult to remove, use a needle to gently break away any LMPA surrounding the embryo.</li>
<li>Place embryos in a plate with methylene blue fish water and raise the embryos to desired stages for analysis (morphological, immunohistochemical, in situ hybridization, etc and microscopic anlaysis).</li>
</ol>

5. Representative Results: 

<img src="/files/ftp_upload/2466/2466fig1.jpg" alt="Figure 1" /> 
Figure 1. Electroporation station. Electrodes were made using sharpened 75&mu;m Tungsten wires and connected by leads to a Protect CUY-21 Edit Square Wave Electroporator. These electrodes were taped to micromanipulators.
<img src="/files/ftp_upload/2466/2466fig2.jpg" alt="Figure 2" /> 
Figure 2. Injection station. All embryos were injected into the right ear for standardization purposes.
<img src="/files/ftp_upload/2466/2466fig3.jpg" alt="Figure 3" /> 
Figure 3. Phalloidin staining of actin filaments with 3 dpf embryos. (A) The 3 dpf embryo injected with control MO has strong staining of phalloidin in the posterior macula (pm). (B) The embryo which was injected with control MO and then electroporated had similar levels of phalloidin staining in pm in the injection only embryos. (C) Injection with mif MOs into the otic vesicle alone did not cause reduction of phalloidin staining, while injection of mif MOs along with electroporation caused a dramatic reduction of phalloidin staining in the pm (D). am, anterior macula.
<img src="/files/ftp_upload/2466/2466fig4.jpg" alt="Figure 4" /> 
Figure 4. Phalloidin staining of actin filaments with 5 dpf larvae. There was no difference between injection only (A) and injection with electroporation when the standard control morpholino was used (B). However, the 5 dpf larvae that were injected with mif morpholinos and electroporated showed reduced phalloidin staining in the posterior macula (pm) (D), though less dramatic than 3 dpf, when compared with the embryo that was injected with mif morpholinos only (C). am, anterior macula.
<img src="/files/ftp_upload/2466/2466fig5.jpg" alt="Figure 5" /> 
Figure 5. Acetylated tubulin staining of embryos at 3 dpf. The embryo in (A) did not receive any injection or electroporation. The embryo in (B) was electroporated but received no injection. Embryos receiving both injection and electroporation (C, D) with mif morpholinos had visibly diminished tubulin staining compared to controls. (pm, posterior macula).
<img src="/files/ftp_upload/2466/2466fig6.jpg" alt="Figure 6" /> 
Figure 6. Acetylated tubulin staining of embryos at 3 dpf with control morpholino. (A) Embryo without any treatment showed strong acetylated tubulin staining in the posterior macula (pm) and extensive innervation. (B) Embryo injected with control morpholino. (C) Embryo treated with electroporation only. (D) Control embryo with control morpholino injected and electroporated has similar levels of acetylated tubulin in the posterior macula and innervation compared to the no treatment control.

<ol>
	<li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus.">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> J Neurosci Methods. 50, 123-127 (1993).</li><li>Suzuki, M., Takamura, Y., Maeno, M., Tochinai, S., Iyaguchi, D., Tanaka, I., Nishihira, J., Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+macrophage+migration+inhibitory+factor+is+essential+for+axis+formation+and+neural+development.">Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development.</a> J Biol Chem. 279, 21406-21414 (2004).</li><li>Barald, K. F., Kelley, M. W., W, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+placode+to+polarization:+new+tunes+in+inner+ear+development.">From placode to polarization: new tunes in inner ear development.</a> Development. 131, 4119-4135 (2004).</li></ol>

The production of the article was sponsored by Gene Tools, LLC who produce reagents mentioned in the video and text.

We have successfully introduced antisense oligonucleotide MOs into the ear of the 24 hpf zebrafish embryo. Embryos that were raised after injection and electroporation were viable. If the embryos survived the electroporation, they grew at normal rates compared to embryos that had received no treatment, as well as embryos that had only been injected and embryos that had only been electroporated.
We observed the effects of mif and mif-like MOs after ear injection and electroporation through labeling with phalloidin and acetylated tubulin. Phalloidin staining of actin filaments in sensory hair cells of the posterior macula indicates that electroporation of mif and mif-like MO at the 24-hpf stage caused changes in hair cell and/or stereocilia numbers when compared to embryos that were only injected with the MOs (Figure 3). This decrease in hair cell numbers is consistent with the findings of Shen et al. (submitted) that MOs to mif and mif-like cause a reduction in the number of hair cells in the saccular macula. The decrease in hair cell numbers in embryos that were injected with MOs and electroporated demonstrate that electroporation may aid in moving the MOs across the cell membrane, thereby increasing the extent of the morphological effects when compared to embryos in which the otic vesicle was only injected. Changes in the morphology of the statoacoustic ganglion were observed through acetylated tubulin staining. The extent of innervation by the statoacoustic ganglion was affected, as embryos that were injected and electroporated show decreased branching of the neurites (Figure 5). There may also be decreased condensation of the ganglionic cells and/or decreased number of ganglion cells, because the acetylated tubulin staining is significantly decreased in embryos that were both injected and electroporated. Because mif has been shown to be critical for nervous system development (Suzuki et al., 2004), the changes seen after electroporation may be the result of increased transfection of the mif and mif-like MO solution into the neuroblast cells.
Electroporation of antisense oligo morpholinos directly into a developing tissue is a useful tool for controlling the expression of a gene during that tissue's development, both spatially and temporally, in the embryo. Electroporation of mif and mif-like MOs into the tissues of the inner ear resulted in abnormal morphology of both the posterior macula and the statoacoustic ganglion. There was a reduction in the number of sensory hair cells in the posterior macula in embryos that had been injected and electroporated in comparison with embryos that had received no treatment or with embryos that had either been only injected or only electroporated. There was also a decrease in the degree of innervation of the posterior sensory patch by the statoacoustic ganglion and the size of the SAG. Electroporation is a valuable method for transfecting macromolecules into specific tissues, with the benefit of observing the primary effects of that particular DNA, RNA, or MO in the desired tissue and discriminating these from secondary effects on other tissues, including the brain, neural crest and periotic mesenchyme on inner ear development (Barald and Kelley, 2004).

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		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>Sutter P-97 electrode puller</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PV820 Pneumatic PicoPump</td>
<td>Equipment</td>
<td>World Precision Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>M3301L Manual Micromanipulator</td>
<td>Equipment</td>
<td>World Precision Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Leica S6D stereomicroscope</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Protect CUY-21 Edit Square Wave Electroporator</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Olympus FV500 confocal microscope</td>
<td>Equipment</td>
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<td>A-M Systems</td>
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<td>03F3172</td>
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<td>Supply</td>
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<td>&nbsp;</td>
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<td>Plastic 100 mm Petri dishes</td>
<td>Supply</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>6 well plastic plates</td>
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			<ol>
<li>MOs: 0.3 mM of a combination of for mif, mif-like MO1, and mif-like MO2 was used. Mif MO is complimentary to the start codon of zebrafish mif mRNA: 5'-acatcggcatgactgcgacagagat-3'. Two MOs were used for mif-like. mif-like MO1 is complimentary to the translational start site: 5'-GTTTCTATATTTATGAACGGCATGA-3', while mif-like MO2 derived from AS sequence at the intron1/exon 2 boundary: 5'-GATTCATCCTCTGAAGACGTAAGCC-3'. Control MO was the scrambled nucleotide sequence from Gene Tools: 5'-CCTCTTACCTCAGTTACAATTTATA- 3'.</li>
<li>methylene blue (0.3 ppm) in fish water</li>
<li> MS222 (tricaine, Sigma; 0.64 mM in fish water)</li>
<li> Low-melting point agarose (LMPA; Roche)</li>
<li> phenol red (Sigma)</li>
<li> Anti-acetylated tubulin (Sigma)</li>
<li> Alexa Fluor&reg; 488 goat anti-mouse second antibody (Molecular probes)</li>
<li>Texas red-phalloidin (Molecular probes)</li>
</ol>		</div>

direct delivery of mif morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

Watch this Scientific Journal Video about Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development at JoVE.com

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

direct-delivery-mif-morpholinos-into-zebrafish-otocyst-injection

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14.2K Views.  University of Wisconsin, Madison. A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Video: Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

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 ...

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 ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...