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>
		<li>Sterilize the laminar flow tissue culture hood by turning on UV light for about 30 min and disinfect the surface with 70% ethanol prior to use. In addition, sterilize all dissection instruments including fine dissection tools and Sylgard-coated dishes for embryo dissection via autoclave or by soaking in 70% ethanol for at least 20 min before use. Pour off 70% ethanol and allow the dishes and instruments to air dry in a clean laminar flow clean bench prior to use.</li>
		<li>Prepare sterile Hank&#8217;s Balanced Salt Solution (HBSS)/(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) mixture by adding 10% of 10x HBSS and 0.5% HEPES and adjust the pH to approximately 7.2 and filter sterilize the final solution and store at 4 &#176;C. Carry out all dissections in chilled HBSS/HEPES solution to better preserve the tissue throughout the procedure.</li>
		<li>Coat glass bottom dishes with basement membrane matrix by adding 5 ml of cold (4 &#176;C) sterile Dulbecco&#8217;s Modified Eagle Medium (DMEM) to a 300 &#181;l aliquot of basement membrane matrix in a sterile 15 ml polypropylene tube. Mix by vortexing the contents and add 150 &#181;l of the basement membrane matrix-DMEM mixture to the center of each culture dish so that it covers the entire culture well present in the center of the dish. Cover the dishes and store in a 37 &#176;C incubator for at least 45 min before use.</li>
		<li>Pour 4-5 ml chilled HBSS into three sterile polystyrene petri dishes and leave closed in the hood.</li>
		<li>Prepare culture medium in a sterile 15 ml polypropylene tube by adding 9 ml of DMEM, 100 &#181;l of N2 supplements, 10 g/ml Cipro and 1 ml of FBS.</li>
	</ol>
	</li>
	<li>Isolation of embryos: 
	NOTE: In this procedure for culturing cochlear explants from embryonic mice, harvest inner ears from embryonic day (E) 13 temporal bones with the day after fertilization being considered as E19. The protocol can also be used with embryonic mouse inner ears beginning from E12 to E18.
	<ol>
		<li>Euthanize a timed-pregnant mouse with CO2 and sacrifice animal by cervical dislocation or appropriate approved protocols. Carry out euthanization away from laminar-flow tissue culture hood to maintain sterility in the tissue culture room.</li>
		<li>Place euthanized mouse on paper towel with abdomen facing up and disinfect abdomen with 70% ethanol.</li>
		<li>Open the abdominal cavity by grabbing skin with curved forceps with one hand and cut epidermis and muscle along the midline with scissors using another hand. Make two perpendicular cuts on both sides and pull out the uterus carefully by lifting up bilateral uterine horns with forceps and detach it from underneath connective tissue using scissors.</li>
		<li>Place the embryonic chain in a petri dish containing chilled dissection solution (HBSS/HEPES mixture) and transfer to the laminar-flow clean bench.</li>
		<li>Remove embryos carefully from placenta and place them into one of the sterile Petri dishes containing dissection solution. If the solution turns bloody, move them to a new Petri dish. 
		NOTE: At this point, embryos can be staged using a Theiler staging guide.</li>
		<li>Decapitate embryos by pinching at the neck region with a pair of clean forceps or scissors and place the heads in a fresh Petri dish containing chilled dissection solution.</li>
	</ol>
	</li>
	<li>Dissection of the inner ears:
	<ol>
		<li>Place one embryo head in a sterile sylgard dish containing cold dissecting</li>
		<li>solution. Working under a dissecting microscope at a magnification of ~1.6X, immobilize the embryo head by placing Minutien pins around or closer to eye region (Figure 1A).</li>
		<li>Using two pairs of sterile forceps carefully remove the skin and open up the skull on the dorsal side along the midline (Figure 1B). Remove brain from cranial cavity and inner ears located within temporal bones can be identified at this stage (Figure 1C, D). The lining of the blood vessel, which is usually present around the inner ears, can be used as a landmark for identifying the inner ear at this stage (Figure 1D).</li>
		<li>Dissect the inner ear from the temporal bones by placing the forceps underneath the tissue and isolating the inner ear from base of the skull and transfer to a new dish containing cold dissecting solution (Figure 1E, F).</li>
	</ol>
	</li>
</ol>
2. Generation of Organ of Corti Explant Cultures
NOTE: At this stage of development, the tissue is cartilaginous and can be easily dissected using forceps.
<ol>
	<li>Orient the inner ear so that ventral side is facing up (Figure 2A) and stabilize the inner ear by gently inserting the sharp end of Minutien pins through the vestibular portion of the inner ear (Figure 2B).</li>
	<li>Using ultrafine forceps cut open the overlying cartilage by making an incision using one end of forceps near the oval window and carefully remove the cartilage from the cochlea (Figure 2C). 
	NOTE: It is important to make sure that forceps are not inserted too deeply into the cartilage as overlying cartilage is sometimes fused with the cochlear duct in which case it is helpful to use closed forceps to release cartilage from the underlying cochlear duct by scraping below the surface of the cartilage. At this stage of development, the cochlear spiral is only three quarters of a turn in length.</li>
	<li>Next, expose the sensory epithelium by placing the forceps at the preferred region of the cochlear duct, either the base or at the very apex, and gently pulling out the roof of the cochlea (Figure 2D). 
	NOTE: The roof of the cochlea must be removed completely as it can mask the sensory epithelium hindering the analysis. 
	NOTE: This procedure must be done gently as it is easy to tear the sensory epithelium.</li>
	<li>As a last step, carefully remove the underlying connective tissue from the exposed sensory epithelium so that the base of the cochlear explant is uniformly flat.</li>
	<li>Isolate the dissected cochlea from the vestibular portion of the inner ear using forceps. This can be performed before or after step 2.4 (Figure 2E).</li>
	<li>Transfer the dissected cochlear sensory epithelium into a basement membrane matrix-coated culture well using a 1.5 mm sterile scooper (Figure 2F).</li>
	<li>Orient the cochlear explant with the lumenal surface of the epithelium facing up and aspirate the basement membrane matrix-DMEM solution to carefully flatten the tissue and replace it with 150 &#181;l of fresh culture medium by gently adding to the dish. Care should be taken to make sure that each explant adheres well to the coated glass coverslip and is not floating in the culture medium. Ensure that the tip of the forceps is pointed up while affixing to the glass well to avoid damaging the cochlear explant.</li>
	<li>Gently transfer the culture dish into a sterile 150 mm culture dish and place in a tissue culture incubator at 37 &#176;C with 5% CO2 for desired period of time, usually 3-6 days in vitro (DIV). After 1 DIV, examine the cultures under a dissecting microscope to make sure that the cochlear explant has adhered well to the culture dish.</li>
	<li>After incubation in vitro for desired lengths of time, cultures are processed for immunohistochemistry. 
	NOTE: The explant cultures usually are incubated for 6 DIV which is equivalent to developmental stage, P0. After incubation in vitro, cultures can be processed for further downstream applications like immunohistochemistry (Figure 3B), RT-PCR, in situ hybridization, and/or western blot.</li>
</ol>
3. Electroporation-mediated Gene Transfer
<ol>
	<li>Setting up for electroporation:
	<ol>
		<li>Sterilize one 100 mm Sylgard-coated glass dish by autoclaving or soaking in 70% ethanol for about 20 min and allow to air dry in a clean bench prior to use.</li>
		<li>Prepare DNA from an expression vector of choice using appropriate maxi- or midi-prep kits. The expression vectors used in this protocol are pIRES2-Atoh1.EGFP and pCLIG-NeuroD1.EGFP. The final concentration of DNA should be at least 1 &#181;g/&#181;l in sterile DNA/RNAse-free water.</li>
	</ol>
	</li>
	<li>Electroporating DNA into embryonic cochlear explants:
	<ol>
		<li>Add 10 &#181;l of plasmid DNA solution to a fresh 100 mm Sylgard coated dish and transfer a fully dissected cochlear explant (obtained from step 2.5) into the DNA solution.</li>
		<li>With lumenal surface of the epithelium facing up, tilt the cochlea slightly so that it&#8217;s perpendicular to the plane of the dish.</li>
		<li>Place the electrode paddles on either side of the cochlea with negative paddle (cathode) towards the sensory epithelium and the positive paddle (anode) located adjacent to the base of the cochlea. 
		NOTE: Since DNA is negatively charged, it migrates from negative to positive electrode and by placing the cathode end of the probe adjacent to the sensory epithelium; the DNA will mostly go through the surface of the sensory epithelium.</li>
		<li>Using electroporator, deliver 9 to 10 pulses of 24 mV, 30 msec pulse duration with the foot pedal switch on the electroporator, and then add 100 &#181;l of warm culture medium to the electroporated cochlea.</li>
		<li>Repeat steps 3.2.1-4 for all the cochlear explants and for DNA of all genes of interest and transfer the electroporated cochleae into the basement membrane matrix-coated dish for plating (step 2.7). Culture all the electroporated cochleae in a 37 &#176;C, 5% CO2 humidified incubator for desired lengths of time which will then be processed for immunocytochemistry. 
		NOTE: The cochlea from an entire litter of CD1 mouse (approximately 8-12 pups) can be isolated, microdissected, electroporated and plated in &#60;4 hr.</li>
	</ol>
	</li>
</ol>
4. Analysis of Cochlear Explant Cultures
NOTE: The cultures are usually incubated for 6 DIV which is equivalent to developmental stage, P0. After incubation in vitro, the cultures are fixed and processed for immunocytochemistry.
<ol>
	<li>After desired length of incubation, remove culture medium and quickly rinse the cochlear explant in phosphate-buffered saline (PBS) and incubate in 4% paraformaldehyde (prepared in PBS) for 15 min at room temperature.</li>
	<li>Wash the fixative by adding PBS and rinse in PBS three times for 15 min.</li>
	<li>Permeabilize using PBS-T (PBS + 0.5% Tween) for 30 min before blocking with PBS-T containing 10% goat serum.</li>
	<li>Incubate in primary antibody solution containing primary antibody in PBS-T with 1% goat serum overnight at 4 &#176;C.</li>
	<li>Wash the cochlea three times at room temperature using PBS-T 15 min each before adding appropriate Alexa-conjugated secondary antibodies.</li>
	<li>Since the cochlear explant is cultured on glass coverslip it can be handled individually by isolating the coverslip from the dish and mounting on a slide. This can be achieved by soaking the culture dish in OS30 solvent for at least 1 hr at room temperature. Using the sterile razor blade, detach the coverslip from the dish and mount on a slide and visualize under a fluorescent microscope.</li>
</ol>

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No conflicts of interest declared.

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><tbody><tr><td>HBSS</td><td>Gibco</td><td>14065-056</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Medium</td><td>Gibco</td><td>12430-054</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10082</td><td></td></tr><tr><td>N-2 Supplement (100x)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Ciprofloxacin hydrochloride</td><td>Cellgro</td><td>61-277-RF</td><td></td></tr><tr><td>Glass Dish 60 mm</td><td>Kimble Chase</td><td>23062-6015/23064-6015</td><td></td></tr><tr><td>Glass Dish 100 mm</td><td>Kimble Chase</td><td>23064-10015/23062-10015</td><td></td></tr><tr><td>Minutien pins</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Pulse generator</td><td>Protech International Inc</td><td>CUY21Vivo-SQ</td><td></td></tr><tr><td>Glass bottom culture dishes</td><td>MatTek</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Matrigel matrix</td><td>BD Biosciences</td><td>356237</td><td></td></tr><tr><td>Culture dish, 60 x 15 mm</td><td>Becton Dickinson</td><td>353037</td><td></td></tr><tr><td>Tissue culture dishes</td><td>Greiner Bio-one</td><td>639160</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OS-30</td><td>Dow Corning</td><td>4021768</td><td></td></tr><tr><td>Fluoromount</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Conical tubes, 15 ml</td><td>Greiner Bio-one</td><td>188261</td><td></td></tr><tr><td>Myosin 6</td><td>Proteus Biosciences Inc</td><td>25-6791</td><td></td></tr><tr><td>Myosin 7a</td><td>Proteus Biosciences Inc</td><td>25-6790</td><td></td></tr><tr><td>TuJ1</td><td>Sigma</td><td>T2200</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 ...

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Developmental Biology

12.8K Views.  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. 

Video: Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

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.

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.

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

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genetics

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Neuroscience

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

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

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

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.

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

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

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

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

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

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

Biology

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

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 ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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