We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.

NOTE: All protocols using live animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved methods for the care and use of the laboratory animals. All the dissections should be performed using sterile technique on a clean laminar flow bench. Gloves and a mask, if desired, should be worn during this procedure.
1. Dissection of the Embryonic Mouse Inner Ear
<ol>
	<li>Setting up for organ of Corti explant cultures:
	<ol>
		...

<ol>
	<li>Sobkowicz, H. M., Bereman, B., Rose, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+development+of+the+organ+of+Corti+in+culture.">Organotypic development of the organ of Corti in culture.</a> J Neurocytol. 4 (5), 543-572 (1975).</li><li>Zheng, J. L., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Math1+induces+robust+production+of+extra+hair+cells+in+postnatal+rat+inner+ears.">Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears.</a> Nat Neurosci. 3 (6), 580-586 (2000).</li><li>Zheng, J. L., Shou, J., Guillemot, F., Kageyama, R., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes1+is+a+negative+regulator+of+inner+ear+hair+cell+differentiation.">Hes1 is a negative regulator of inner ear hair cell differentiation.</a> Development. 127 (21), 4551-4560 (2000).</li><li>Woods, C., Montcouquiol, M., 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=Math1+regulates+development+of+the+sensory+epithelium+in+the+mammalian+cochlea.">Math1 regulates development of the sensory epithelium in the mammalian cochlea.</a> Nat Neurosci. 7 (12), 1310-1318 (2004).</li><li>Jones, J. M., Montcouquiol, M., Dabdoub, A., Woods, C., 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=Inhibitors+of+differentiation+and+DNA+binding+(Ids)+regulate+Math1+and+hair+cell+formation+during+the+development+of+the+organ+of+Corti.">Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti.</a> J Neurosci. 26 (2), 550-558 (2006).</li><li>Dabdoub, A., 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=Sox2+signaling+in+prosensory+domain+specification+and+subsequent+hair+cell+differentiation+in+the+developing+cochlea.">Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (47), 18396-18401 (2008).</li><li>Doetzlhofer, A., Basch, M. L., Ohyama, T., Gessler, M., Groves, A. K., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hey2+regulation+by+FGF+provides+a+Notch-independent+mechanism+of+maintaining+pillar+cell+fate+in+the+organ+of+Corti.">Hey2 regulation by FGF provides a Notch-independent mechanism of maintaining pillar cell fate in the organ of Corti.</a> Dev Cell. 16 (1), 58-69 (2009).</li><li>Puligilla, C., Dabdoub, A., Brenowitz, S. D., 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=Sox2+induces+neuronal+formation+in+the+developing+cochlea.">Sox2 induces neuronal formation in the developing cochlea.</a> J Neurosci. 20 (2), 714-722 (2010).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1995).</li><li>Sher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+and+postnatal+development+of+the+inner+ear+of+the+mouse.">The embryonic and postnatal development of the inner ear of the mouse.</a> Acta Otolaryngol Suppl. 285, 1-77 (1971).</li><li>Torres, M., 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+development+of+the+vertebrate+inner+ear.">The development of the vertebrate inner ear.</a> Mech Dev. 71 (1-2), 5-21 (1998).</li><li>Brown, S. T., Martin, K., Groves, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+inner+ear+induction.">Molecular basis of inner ear induction.</a> Curr Top Dev Biol. 57, 115-119 (2003).</li><li>Puligilla, C., 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=Building+the+world&#8217;s+best+hearing+aid;+regulation+of+cell+fate+in+the+cochlea.">Building the world&#8217;s best hearing aid; regulation of cell fate in the cochlea.</a> Curr Opin Genet Dev. 19 (4), 368-373 (2009).</li><li>Wu, D. K., 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=Molecular+mechanisms+of+inner+ear+development.">Molecular mechanisms of inner ear development.</a> Cold Spring Harb Perspect Biol. 4 (8), a008409 (2012).</li><li>Holt, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral-mediated+gene+transfer+to+study+the+molecular+physiology+of+the+mammalian+inner+ear.">Viral-mediated gene transfer to study the molecular physiology of the mammalian inner ear.</a> Audiol Neurootol. 7 (3), 157-160 (2002).</li><li>Stone, I. M., Lurie, D. I., Kelley, M. W., Poulsen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+transfer+to+hair+cells+and+support+cells+of+the+murine+cochlea.">Adeno-associated virus-mediated gene transfer to hair cells and support cells of the murine cochlea.</a> Mol Ther. 11 (6), 843-848 (2005).</li></ol>

All the cells within the membranous labyrinth of the mouse inner ear including sensory, nonsensory and the spiral ganglion neurons are all derived from placodally-derived otocyst located adjacent to the hindbrain of the ectoderm, around E810-14. At E11, the ventral region of the otocyst extends to form the cochlear duct and as development continues, a group of epithelial cells within the cochlea, as well as in other regions of the otocyst, become specified as prosensory patches that will subsequently give rise...

The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis. 
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results. 
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">HBSS</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:161px;">14065-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">HEPES</td>
			<td>Gibco</td>
			<td style="width:161px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Dulbecco&#8217;s Modified Medium</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">12430-054</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Fetal Bovine Serum</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">10082</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">N-2 Supplement (100X)</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Ciprofloxacin Hydrochloride</td>
			<td dir="LTR" style="width:192px;">Cellgro</td>
			<td dir="LTR" style="width:161px;">61-277-RF</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Glass Dish 60mm</td>
			<td dir="LTR" style="width:192px;">Kimble Chase</td>
			<td dir="LTR" style="width:161px;">23062-6015/23064-6015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Glass Dish 100 mm</td>
			<td dir="LTR">Kimble Chase</td>
			<td style="width:161px;">23064-10015/23062-10015</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Minutien Pins</td>
			<td dir="LTR" style="width:192px;">Fine Science Tools</td>
			<td style="width:161px;">26002-15</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Dumont # 5 Forceps</td>
			<td dir="LTR" style="width:192px;">Fine Science Tools</td>
			<td dir="LTR" style="width:161px;">11251-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Pulse Generator&#160;</td>
			<td dir="LTR">Protech International Inc</td>
			<td style="width:161px;">CUY21Vivo-SQ</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Glass Bottom Culture Dishes</td>
			<td dir="LTR" style="width:192px;">MatTek</td>
			<td dir="LTR" style="width:161px;">P35G-0-10-C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Matrigel Matrix</td>
			<td dir="LTR" style="width:192px;">BD Biosciences</td>
			<td dir="LTR" style="width:161px;">356237</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Culture Dish, 60 X 15 mm</td>
			<td dir="LTR" style="width:192px;">Becton Dickinson</td>
			<td style="width:161px;">353037</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Tissue&#160; Culture Dishes</td>
			<td dir="LTR" style="width:192px;">Greiner Bio-one</td>
			<td style="width:161px;">639160</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Phosphate Buffered Saline</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">OS-30</td>
			<td dir="LTR" style="width:192px;">Dow Corning</td>
			<td dir="LTR" style="width:161px;">4021768</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Fluoromount</td>
			<td dir="LTR" style="width:192px;">Southern Biotech</td>
			<td dir="LTR" style="width:161px;">0100-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Conical Tubes, 15ml</td>
			<td style="width:192px;">Greiner Bio-one</td>
			<td style="width:161px;">188261</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Myosin 6</td>
			<td>Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6791</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Myosin 7a</td>
			<td style="width:192px;">Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6790</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">TuJ1</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:161px;">T2200</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest...

Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by 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.

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

culture-embryonic-mouse-cochlear-explants-gene-transfer

Research

JoVE Journal

Developmental Biology

12.8K vistas.  Medical University of South Carolina, College of Medicine. 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. 