Name: Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation (Video) | JoVE Journal
Uploaded: 2016-04-17T15:00:00.000+00:00
Duration: 6 min 45 s
Description: Learn in ovo micro-electroporation for genetic manipulation of the developing chicken auditory organ from embryonic day four. This technique enables precise study of auditory progenitor cell proliferation and differentiation.

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5 minutes

We thank Dr. Doris K. Wu for expression plasmids and in situ probes, the Johns Hopkins University Center for Sensory Biology imaging facility and the Center for Hearing and Balance. This work was supported by NIDCD Grant T32 DC000023 to L.E.

The eggs and unhatched embryos are cared for and treated ethically&#160;and humanely. All protocols for unhatched embryo use were approved by the Animal Care and Use Committee at the Johns Hopkins School of Medicine, Baltimore, Maryland.
1. Eggs and Preparation of Expression Constructs
<ol>
	<li>Eggs:
	<ol>
		<li>Incubate 3 - 4 dozen fertilized chicken eggs (Gallus gallus) lying flat on their sides at 37 &#176;C.
		<ol>
			<li>After 24 hr&#160;of incubation, pull 3 cc of albumen with a syringe from the round back end of the egg and seal the hole with clear tape. This creates an air chamber between the embryo and the eggshell, allowing for ease of access to the embryo without the embryo sticking to the shell when cutting the window for access on top of the egg 19.</li>
			<li>At 117 hr&#160;of incubation time, stage the embryos according to Hamburger and Hamilton 20 developmental stages at E4 (HH24-25) (See Figure 1D).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Expression Constructs:
	<ol>
		<li>Prepare the plasmid DNA using reagents and the protocol provided by the manufacturer of a commercially available preparation kit. At the final step of preparation, elute the DNA into 500 &#181;l TE (Tris-EDTA) buffer provided in the kit into a 1.5 ml tube.</li>
		<li>Ethanol precipitate the DNA to concentrate it by adding 50 &#181;l of 3 M Sodium Acetate and 1 ml of 100% Ethanol to the tube. Place the tube at -20 &#176;C O/N.</li>
		<li>Centrifuge the tube for 30 min at 14,000 rpm at 4 &#176;C. Remove the supernatant and air-dry the pellet for 10 min at RT. Dissolve the pellet in 15 &#181;l TE buffer, which should yield a concentration of ~4 &#181;g/&#181;l. 
		NOTE: The control expression constructs used here are pMES-IRES-GFP10, which is driven by a chicken &#946;-actin promoter, or pCI-H2B-IRES-RFP10, which is driven by the CMV promoter.</li>
	</ol>
	</li>
</ol>
2. Chicken In Ovo&#160;Micro-electroporation and In Ovo&#160;Cell Proliferation Assay
<ol>
	<li>In Ovo&#160;Micro-injection of Expression Construct and Micro-electroporation:
	<ol>
		<li>Pull the glass capillary tubes into fine needles using a micro-pipette puller with the following optimized parameters for a 2.5 x 2.5 mm Box Platinum Heating Filament: Pressure = 500, Heat = 600 &#176;C, Pull = 64, Velocity = 105, and Time = 150 msec (see Table 1).</li>
		<li>Set up the Left- and Right-handed micro-manipulator stages flanking the microscope (see Figure 1A). The Right-handed micro-manipulator holds the glass capillary needle and the Left-handed micro-manipulator holds the electrodes (Figure 1A).</li>
		<li>Prepare the finely pulled glass capillary needle for micro-injection by cutting the tip of the needle with a forceps while working under the microscope.</li>
		<li>Fill the needle manually by hand using the micro-manipulator with 2.5 &#181;l of plasmid DNA tinted with 0.1% Fast Green while working under the microscope.</li>
		<li>Place the egg lying on its side within a mold/egg holding device (see Figure 1A). Cut a round window on top of the egg using scissors 19 (Figure 1A).</li>
		<li>While working under the microscope, carefully open the two membranes overlaying the embryo using forceps.</li>
		<li>While working under the microscope, deliver approximately ~0.5 &#181;l of plasmid DNA to the right otic vesicle lumen by micro-injection manually using the right hand by using the micro-manipulator and by micro-electroporation.
		<ol>
			<li>To do so, micro-inject the right otic vesicle lumen with the DNA with the Right-hand micro-manipulator (Figure 1B-D) while at the same time using the left hand holding a forcep to steady the head of the embryo, because the head of the embryo dips at stage E4. Do not micro-inject past the otic vesicle lumen, because this will damage the otic vesicle.</li>
			<li>Immediately after micro-injection, while working under the microscope, use the Left-hand micro-manipulator to place the positive (anode) 2 mm platinum electrode anterior-ventral to the right otic vesicle and the negative 2 mm electrode (cathode) in parallel 1 mm apart (see Figure 1 C). Deliver 4 pulses at 12 V with 100 msec duration and 200 msec spacing. The left otic vesicle serves as an internal untreated control. 
			NOTE: Clean the electrodes in between electroporations with a cotton-tipped swab dipped in 1x PBS.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In Ovo&#160;Cell Proliferation Assay:
	<ol>
		<li>Immediately after electroporation add 50 &#956;l of 0.25 mg/ml EdU in 1x PBS by manually dropping the 50 &#956;l EdU solution onto the whole embryo in ovo using a pipette. Seal the eggs with tape and return to the incubator for 18 -&#160;96 hr.</li>
		<li>Carefully remove the tape and check the embryos for fluorescent GFP or RFP signal within the otic vesicle after 18 - 24 hr&#160;using a standard fluorescent microscope (see Figure 1 E-E&#39;). If fluorescent signal is present, at this point harvest the embryos for analyses or reseal with tape and return it to the incubator for harvesting and analyses at later stages (see Figure 1 F-P&#39;&#39;). 
		NOTE: Expression of GFP signal with the pMES-IRES-GFP construct is evident 6 - 7 hr&#160;after electroporation 10.</li>
	</ol>
	</li>
</ol>
3. Embryo Harvesting and Tissue Processing for RNA In Situ hybridization and Immunohistochemistry
<ol>
	<li>Harvest the embryos using forceps. Rinse the embryo in cold 1x PBS. Remove the heads of the embryos, by cutting the head by its neck using forceps, and place the heads into 4% paraformaldehyde O/N at 4 &#176;C. Puncture the brain using a forcep to allow penetration of the 4% paraformaldehyde inside the head.</li>
	<li>Dehydrate the heads in 30% sucrose (in 1x PBS) O/N at 4 &#176;C.</li>
	<li>Mount the heads in cryoprotective medium by rapid freezing in a slurry of dry ice and methylbutane (2-Methylbutane). Alternatively, rapid freeze the heads in cryoprotective medium with liquid nitrogen. Store the mounted heads at -80 &#176;C.</li>
	<li>Cryosection the heads into 12 &#181;m thick tissue sections and collect all the inner ear-containing tissue sections onto superfrosted microscope glass slides.</li>
	<li>Perform standard RNA in situ hybridization and immunohistochemistry as previously described 4,21, and analyze for EdU cell proliferation following the protocol provided by the manufacturer of the kit. 
	NOTE: Hair cells can be identified using rabbit polyclonal &#945;-MyosinVIIa (Myo7a) antibody (1:1,000, Proteus Biosciences) and fluorescently-labeled secondary antibody (1:250, Goat anti-Rabbit AlexaFluor 488; Invitrogen).</li>
</ol>
4. Image Capture and Processing
<ol>
	<li>Take images with digital imaging equipment. Image immunohistochemistry results with a standard fluorescent microscope and results of the in situ using a standard wide-field microscope (ranging from 5x to 40x in magnification).</li>
	<li>Process the images using image processing software.</li>
</ol>

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<li>Freeman, S., Chrysostomou, E., Kawakami, K., Takahashi, Y., Daudet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tol2-mediated+gene+transfer+and+in+ovo+electroporation+of+the+otic+placode%3A+a+powerful+and+versatile+approach+for+investigating+embryonic+development+and+regeneration+of+the+chicken+inner+ear%5BTitle%5D)+AND+%22Methods+in+molecular+biology%22%5BJournal%5D)">Tol2-mediated gene transfer and in ovo electroporation of the otic placode: a powerful and versatile approach for investigating embryonic development and regeneration of the chicken inner ear.</a> Methods in molecular biology. 916, 127-139 (2012).</li>
<li>Kamaid, A., Neves, J., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Id+gene+regulation+and+function+in+the+prosensory+domains+of+the+chicken+inner+ear%3A+a+link+between+Bmp+signaling+and+Atoh1%5BTitle%5D)+AND+%22The+Journal+of+neuroscience+%3A+the+official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Id gene regulation and function in the prosensory domains of the chicken inner ear: a link between Bmp signaling and Atoh1.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 30, 11426-11434 (2010).</li>
<li>Neves, J., Kamaid, A., Alsina, B., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+expression+of+Sox2+and+Sox3+in+neuronal+and+sensory+progenitors+of+the+developing+inner+ear+of+the+chick%5BTitle%5D)+AND+%22The+Journal+of+comparative+neurology%22%5BJournal%5D)">Differential expression of Sox2 and Sox3 in neuronal and sensory progenitors of the developing inner ear of the chick.</a> The Journal of comparative neurology. 503, 487-500 (2007).</li>
<li>Neves, J., Uchikawa, M., Bigas, A., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+prosensory+function+of+Sox2+in+the+chicken+inner+ear+relies+on+the+direct+regulation+of+Atoh1%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The prosensory function of Sox2 in the chicken inner ear relies on the direct regulation of Atoh1.</a> PLoS One. 7, 30871(2012).</li>
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<li>Wu, C. Y., Hooper, R. M., Han, K., Taneyhill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Migratory+neural+crest+cell+alphaN-catenin+impacts+chick+trigeminal+ganglia+formation%5BTitle%5D)+AND+%22Developmental+biology%22%5BJournal%5D)">Migratory neural crest cell alphaN-catenin impacts chick trigeminal ganglia formation.</a> Developmental biology. 392, 295-307 (2014).</li>
<li>Kumar, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+non-small+cell+lung+tumor+development+by+the+let-7+microRNA+family%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Suppression of non-small cell lung tumor development by the let-7 microRNA family.</a> Proc Natl Acad Sci U S A. 105, 3903-3908 (2008).</li>
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The authors have nothing to disclose.

The here described method of in ovo micro-electroporation is optimized for gene transfer into the developing auditory organ. It is compatible with plasmid DNA-based expression vectors typically used to manipulate gene function/expression. The timing of electroporation at E4 is optimal for investigations that focus on HC development in the auditory organ. The most critical steps are micro-injecting the DNA into the otic vesicle lumen without going too deep with the needle and damaging the otic vesicle (see <stron...

Despite differences in morphology and cellular patterning between the mammalian and avian auditory sensory organ, the molecular factors and pathways responsible for sensory HC development are thought to be evolutionarily conserved 1-3. The basilar papilla, which houses the auditory HCs and their surrounding SCs, develops as an outpocketing of the inner ear otocyst. Early on a pool of HC and SC progenitors is specified within the otic placode/otic cup neural-sensory competent domain (NSD). Fate-mapping data provide evidence that neuronal and sensory lineages are linked and arise from the NSD located in the antero-ventral region of the otic cup or otocyst in mice and chicken 4,5. First, neuroblasts delaminate from the NSD to give rise to the neurons of the auditory-vestibular ganglion, which eventually split into auditory and vestibular ganglia. These neurons innervate the sensory HCs of the inner ear and nuclei in the brainstem. The cells that remain in the NSD are thought to give rise to various sensory patches, including the auditory sensory organ, consisting of the mechano-transducing sensory HCs and their associated SCs.
In both the chick and murine, auditory organ sensory progenitor cell-cycle exit and HC differentiation occur in opposing gradients. In chick, auditory progenitor cell-cycle exit starts around ~E5 and progresses from the base to the apex, and from the center to the periphery 6. One day later, HC differentiation starts in the apex and progresses to the base 7. Studying the development of the inner ear in chicken provides many technical advantages as functional mechanisms can be investigated with relative ease in ovo as opposed to manipulating embryos in utero in other model systems, which requires complex surgeries. The method described here uses in ovo micro-electroporation to target genes of interest in the presumptive basilar papilla area anterior-ventrally in the otic vesicle specifically at E4.
Electroporation in ovo is a technique that is well established and commonly used 8-14. The principle of the electroporation technique is based on the fact that nucleotides (e.g., plasmid DNA, synthetic DNA, or RNA oligonucleotides) are negatively charged. The DNA is injected into the tissue of interest. When an electric current is placed across the tissue, the current opens up transient pores in the cell walls and allows for the uptake of the DNA, as the negatively charged DNA flows toward the positive electrode (anode). For gain-of-function experiments, genes of interest are commonly subcloned into expression plasmids that contain appropriate expression cassettes for green fluorescent protein (GFP) or red fluorescent protein (RFP). For loss-of-function experiments plasmid-based dominant negative repressor constructs, or RNAi, or morpholino constructs are commonly used 15,16. To inhibit microRNA (miRNA) function miRNAs sponge constructs, which are typically plasmid based, can be used 17. The here described method allows for investigating the molecular mechanisms that control proliferation and differentiation in the auditory organ, as the in ovo micro-electroporation method can be easily combined with in ovo cell proliferation assay by adding EdU to the whole embryo.
The electroporation and addition of EdU is performed at E4 in the otic vesicle, which is one day before the onset of cell-cycle exit in the auditory organ. Analysis of the auditory organ is typically performed 18 - 96 hr&#160;after electroporation at E5 (onset of sensory progenitor cell-cycle exit), E6 (onset of HC differentiation), and E7 (during HC differentiation); and later up to ~E9 (end of HC differentiation). This electroporation method of gene transfer is transient lasting approximately ~4 days, because the genes of interest do not integrate into the genome, but the method is applicable for use with appropriate plasmid DNA-based expression vectors, which do have the ability to integrate into the genome, such as Tol2-mediated gene transfer 11. With this method robust GFP or RFP expression lasts ~4 days in the cochlea, after which point the signals fade, yet provide an ample time window to study the intricate development of the auditory organ. This in ovo gene transfer method is novel and allows to specifically target the presumptive auditory organ at E4, which is optimal for investigations that focus on HC development in the auditory organ. It is a good addition to alternative methods, which electroporate at much younger developmental stages 11,12 or compared to the use of in vitro basilar papillae explant cultures 18.

<table><tbody><tr><td>Sterile 1x PBS pH 7.4</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Fast Green FCF Powder</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>EdU Powder</td><td>Invitrogen</td><td>E10187</td><td></td></tr><tr><td>Click-IT EdU Alexa Fluor 555 Imaging Kit</td><td>Invitrogen</td><td>C10338</td><td></td></tr><tr><td>HiSpeed Plasmid Midi Kit (25)</td><td>Qiagen</td><td>12643</td><td></td></tr><tr><td>ECM 830 ElectroSquarePorator&amp;nbsp;</td><td>BTX Harvard Apparatus</td><td></td><td></td></tr><tr><td>Banana to Micrograbber Cable Kit</td><td>BTX Harvard Apparatus</td><td>45-0216</td><td></td></tr><tr><td>Right Handed &amp;amp; Left Handed Micromanipulators</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>M3301R &amp;amp; M3301L</td><td></td></tr><tr><td>Two 12 mm Magnetic Holding Device Stages with 7 inch vertical posts</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>M10</td><td></td></tr><tr><td>Metal Steel Base Plate 12x24 inch</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>5479</td><td></td></tr><tr><td>Scissors for Eggs 12 cm long curved, 12 mm extrafine blades; Spring Scissors</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>14120</td><td></td></tr><tr><td>Micropippette Puller for pulling needles</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>2.5x2.5 mm Box Platinum Heating Filament</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Glass Capillary Tubes/Needles/No Fiber/Borosil 1 mm</td><td>FHC</td><td>27-30-0</td><td></td></tr><tr><td>Hamilton Glass Syringe 100 &amp;mu;l</td><td>Hamilton</td><td>80601 Model 710LT</td><td></td></tr><tr><td>Mineral Oil (Heavy) for Hamilton Glass Syringe</td><td>Fisher Scientific</td><td>O122-1</td><td></td></tr><tr><td>Polyethylene Tubing for connecting the Glass Syringe and Glass Cappillary Needles/ Non Toxic</td><td>Becton Dickinson and Company (BD)</td><td>427420 Intramedic Clay Adams Brand</td><td></td></tr><tr><td>3 cc Disposable Syringes</td><td>Becton Dickinson and Company (BD)</td><td>309657</td><td></td></tr><tr><td>Disposable 21 Gauge Needles</td><td>Becton Dickinson and Company (BD)</td><td>305122</td><td></td></tr><tr><td>One pair of 2 mm Platinum Electrodes</td><td>Bulldog Bio. / Nepagene</td><td>CUY611P3-2</td><td></td></tr><tr><td>Electrode Holder</td><td>Bulldog Bio. / Nepagene</td><td>CUY580</td><td></td></tr><tr><td>One pair of Dumont fine forceps number 5</td><td>Fine Science Tools (FST)</td><td></td><td></td></tr><tr><td>Matte finish invisible tape for sealing eggs</td><td>Office Depot</td><td>520-928</td><td></td></tr><tr><td>Cotton-Tipped Swabs</td><td>Fisher Scientific</td><td>23-400-101</td><td></td></tr><tr><td>Sterile filter tips</td><td></td><td></td><td></td></tr></tbody></table>

gene transfer into the chicken auditory organ by in ovo micro-electroporation

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

In this method paper plasmid DNA consisting of green fluorescent protein (GFP) expression cassettes was targeted into the developing chicken basilar papilla (BP) with optimized parameters of 12 V and 4 pulses with 100 msec pulse duration and intervals of 200 msec, yielding a ~50% embryo survival rate and efficiency of plasmid-DNA targeting into the BP. Fluorescent imaging of native GFP expression in developing embryos showed this method of electroporation preferentially targets the anteri...

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

The overall goal of this method is to perform genetic manipulations in the developing chicken auditory organ from embryonic day four, onward. This is significant because of the difficulty of working with the advanced developmental stage of the chicken embryo. The main advantage of this technique is that it specifically targets the developing auditory organ in ovo with relative ease compared to other techniques which require complex in utero surgeries.This method can help answer key questions of auditory sensory development. Including how the spatial and temporal pattern of sensory progenital cell proliferation and herital differentiation are coordinated. To begin this procedure attach the glass capillary needle using the tubing connected to the glass syringe which is placed onto the right-hand micromanipulator, and place the electrodes onto the left-hand micromanipulator.Under the microscope, cut the needle tip using a pair of forceps. Next, place 2.5 microliters of plasma DNA tinted with 0.1%fast green into a dish. Fill the needle with plasma DNA using the plunger of the syringe attached to the right-hand micromanipulator.Place an egg on its side in a holding device. Then, cut a round window on top of the egg using scissors. After that, place the egg in the holding device under the microscope.Carefully open the two membranes overlaying the embryo using a pair of forceps to expose the right otic vesicle. While stabilizing the head of the embryo with forceps using the left hand, deliver approximately 0.5 microliters of plasma DNA with the right-hand micromanipulator. Be careful not to inject past the otic vesicle lumen because this will damage the otic vesicle.Microinjecting DNA into the otic vesicle lumen without damaging the otic vesicle is critical and it requires the use of both hands. Immediately after microinjection, use the left-hand micromanipulator to place the two millimeter platinum positive and negative electrode set anterior ventral to the right otic vesicle, and deliver four pulses at 12 volts by operating the electroporador. The precise placement of electrodes onto the anterior ventral presumptive auditory organ area is important and ensures that the developing auditory organ is targeted.Immediately after electroporation, add 50 microliters of EdU onto the whole embryo in ovo. Next, seal the egg with tape. Return all the manipulated and sealed eggs to the incubator for 18 to 96 hours.After 18 to 24 hours, carefully remove the tape. Check the embryos for fluorescent GFP or RFP signal within the otic vesicle using a standard fluorescent microscope. If fluorescent signal is present, harvest the embryos for analyses or re-seal them with tape and return them to the incubator for harvesting and analyses at later stages.RNA in situ hybridization was used to determine whether electroporation successfully targeted the auditory portion of the developing inner ear. Within the developing BP, sensory progenitors were identified by their expression of Sox2 and Lunatic Fringe. These images demonstrate that sensory progenitors throughout the developing BP at the base and apex were successfully targeted and expressed GFP after 68 hours of electroporation.In addition, GFP was expressed outside the sensory domain in non-sensory epithelial cells and in the auditory ganglion marked by Neurod1 expression. To determine the proliferative behavior of sensory progenitors, EdU, a thymidine analogue, was added at the time of electroporation at E four, and four days later, EdU incorporation in the developing BP was analyzed in tissue sections. Immunolabeling for the hair cell-specific protein, Myosin7a, was used to identify differentiating hair cells in the developing BP.In the apical portion of the BP, Myo7a positive hair cells were frequently EdU positive.Whereas in the base, only few Myo7a positive hair cells had incorporated EdU, suggesting that at the time of EdU addition at E four, sensory progenitors in the base are already largely postmitotic. Once mastered, this technique can be completed in five to eight minutes per egg. While performing this procedure, it is important to use freshly prepared and sterile reagents to ensure the survival of the embryos.Following this procedure, the method parameters can be modified to study other phenomena in the developing inner ear, such as the development of vestibular organs and inner ear neurogenesis. This technique paves the way for researchers in the inner ear field to specifically study the relatively late developing chicken auditory organ from embryonic day four, onward. After watching this video, you should have a good understanding of how to appropriately handle the chicken embryo, conducting microinjection and electroporation, and conduct the in ovo cell proliferation assay.

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Research

JoVE Journal

Developmental Biology

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo.  Common applications of this technique include functional analysis of genes and putative enhancer elements.  This video demonstrates neural tube electroporation in HH 10 chick embryos.  Injection technique and proper egg handling are discussed.

In Ovo Electroporations of HH Stage 10 Chicken Embryos

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

In Ovo Electroporation in Embryonic Chick Retina

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

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.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear.  We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

In Ovo Electroporation in the Chicken Auditory Brainstem

We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Learn in ovo micro-electroporation for genetic manipulation of the developing chicken auditory organ from embryonic day four. This technique enables precise study of auditory progenitor cell proliferation and differentiation.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

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