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. 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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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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. ...

Research

JoVE Journal

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

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In Ovo Electroporation in the Chicken Auditory Brainstem

In Ovo Electroporation in the Chicken Auditory Brainstem

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

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