Name: gene transfer into the chicken auditory organ by in ovo micro-electroporation
Uploaded: 2016-04-17T15:00:00
Description: Watch this Scientific Journal Video about Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation at JoVE.com

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 ...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+inner+ear.">Development of the inner ear.</a> Current opinion in genetics &amp; development. 32, 112-118 (2015).</li><li>Raft, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-regulation+of+Ngn1+and+Math1+coordinates+the+production+of+neurons+and+sensory+hair+cells+during+inner+ear+development.">Cross-regulation of Ngn1 and Math1 coordinates the production of neurons and sensory hair cells during inner ear development.</a> Development. 134, 4405-4415 (2007).</li><li>Satoh, T., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+the+relationships+between+mechanosensory+cells+and+the+neurons+that+innervate+them+in+the+chicken+ear.">Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear.</a> Development. 132, 1687-1697 (2005).</li><li>Katayama, A., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+production+in+the+chicken+cochlea.">Cell production in the chicken cochlea.</a> The Journal of comparative neurology. 281, 129-135 (1989).</li><li>Cotanche, D. A., Sulik, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+stereociliary+bundles+in+the+cochlear+duct+of+chick+embryos.">The development of stereociliary bundles in the cochlear duct of chick embryos.</a> Brain Res. 318, 181-193 (1984).</li><li>Alsina, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signaling+is+required+for+determination+of+otic+neuroblasts+in+the+chick+embryo.">FGF signaling is required for determination of otic neuroblasts in the chick embryo.</a> Developmental biology. 267, 119-134 (2004).</li><li>Chang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp4+is+essential+for+the+formation+of+the+vestibular+apparatus+that+detects+angular+head+movements.">Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements.</a> PLoS genetics. 4, e1000050 (2008).</li><li>Evsen, L., Sugahara, S., Uchikawa, M., Kondoh, H., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+neurogenesis+in+the+inner+ear+requires+inhibition+of+Sox2+transcription+by+neurogenin1+and+neurod1.">Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by neurogenin1 and neurod1.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33, 3879-3890 (2013).</li><li>Freeman, S., Chrysostomou, E., Kawakami, K., Takahashi, Y., Daudet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Id+gene+regulation+and+function+in+the+prosensory+domains+of+the+chicken+inner+ear:+a+link+between+Bmp+signaling+and+Atoh1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+Sox2+and+Sox3+in+neuronal+and+sensory+progenitors+of+the+developing+inner+ear+of+the+chick.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prosensory+function+of+Sox2+in+the+chicken+inner+ear+relies+on+the+direct+regulation+of+Atoh1.">The prosensory function of Sox2 in the chicken inner ear relies on the direct regulation of Atoh1.</a> PLoS One. 7, 30871 (2012).</li><li>Das, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+system+for+RNA+interference+in+the+chicken+using+a+modified+microRNA+operon.">A robust system for RNA interference in the chicken using a modified microRNA operon.</a> Developmental biology. 294, 554-563 (2006).</li><li>Wu, C. Y., Hooper, R. M., Han, K., Taneyhill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migratory+neural+crest+cell+alphaN-catenin+impacts+chick+trigeminal+ganglia+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+non-small+cell+lung+tumor+development+by+the+let-7+microRNA+family.">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><li>Jacques, B. E., Dabdoub, A., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+signaling+regulates+development+and+transdifferentiation+of+hair+cells+and+supporting+cells+in+the+basilar+papilla.">Fgf signaling regulates development and transdifferentiation of hair cells and supporting cells in the basilar papilla.</a> Hearing research. 289, 27-39 (2012).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> Journal of visualized experiments: JoVE. , e306 (2007).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of morphology. 88, 49-92 (1951).</li><li>Oh, S. H., Johnson, R., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+bone+morphogenetic+proteins+in+the+developing+vestibular+and+auditory+sensory+organs.">Differential expression of bone morphogenetic proteins in the developing vestibular and auditory sensory organs.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 16, 6463-6475 (1996).</li><li>Bell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+segregation+of+auditory+and+vestibular+neurons+in+the+otic+placode.">Spatial and temporal segregation of auditory and vestibular neurons in the otic placode.</a> Developmental biology. 322, 109-120 (2008).</li></ol>

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.

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			<td height="21" style="height:21px;width:621px;">Sterile 1x PBS pH 7.4</td>
			<td style="width:249px;">gibco</td>
			<td style="width:259px;">10010-023</td>
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			<td height="21" style="height:21px;">Fast Green FCF Powder</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
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			<td height="21" style="height:21px;">EdU Powder</td>
			<td>Invitrogen</td>
			<td>E10187</td>
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			<td height="21" style="height:21px;">Click-IT EdU Alexa Fluor 555 Imaging Kit</td>
			<td>Invitrogen</td>
			<td>C10338</td>
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		<tr height="21">
			<td height="21" style="height:21px;">HiSpeed Plasmid Midi Kit (25)</td>
			<td>Qiagen</td>
			<td align="right">12643</td>
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			<td height="21" style="height:21px;">ECM 830 ElectroSquarePorator&#160;</td>
			<td>BTX Harvard Apparatus</td>
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			<td height="21" style="height:21px;">Banana to Micrograbber Cable Kit</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0216</td>
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			<td height="21" style="height:21px;">Right Handed &#38; Left Handed Micromanipulators</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td>M3301R &#38; M3301L</td>
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			<td height="21" style="height:21px;">Two 12 mm Magnetic Holding Device Stages with 7 inch vertical posts</td>
			<td>World Precision Instruments Inc.&#160;</td>
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			<td>World Precision Instruments Inc.&#160;</td>
			<td align="right">14120</td>
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			<td height="21" style="height:21px;">Micropippette Puller for pulling needles</td>
			<td>Sutter Instrument Co.</td>
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			<td>Sutter Instrument Co.</td>
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			<td height="21" style="height:21px;">Glass Capillary Tubes/Needles/No Fiber/Borosil 1 mm</td>
			<td>FHC</td>
			<td>27-30-0</td>
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			<td>Hamilton</td>
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			<td height="21" style="height:21px;">Mineral Oil (Heavy) for Hamilton Glass Syringe</td>
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			<td height="21" style="height:21px;">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>
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			<td>Becton Dickinson and Company (BD)</td>
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			<td height="21" style="height:21px;">Disposable 21 Gauge Needles</td>
			<td>Becton Dickinson and Company (BD)</td>
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			<td height="21" style="height:21px;">One pair of 2 mm Platinum Electrodes</td>
			<td>Bulldog Bio. / Nepagene</td>
			<td>CUY611P3-2</td>
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			<td height="21" style="height:21px;">Electrode Holder</td>
			<td>Bulldog Bio. / Nepagene</td>
			<td>CUY580</td>
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			<td height="21" style="height:21px;">One pair of Dumont fine forceps number 5</td>
			<td>Fine Science Tools (FST)</td>
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			<td height="21" style="height:21px;">Sterile filter tips</td>
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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&#160;DNA consisting of green fluorescent protein (GFP) expression cassettes was targeted into the developing chicken basilar papilla (BP) with optimized parameters of 12 V&#160;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 ...

Watch this Scientific Journal Video about Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation at JoVE.com

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

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.

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 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

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

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.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

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.

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

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

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

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

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.

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...