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

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

<ol>
	<li>Bissonnette, J. P., 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=Standard+atlas+of+the+gross+anatomy+of+the+developing+inner+ear+of+the+chicken.">Standard atlas of the gross anatomy of the developing inner ear of the chicken.</a> The Journal of comparative neurology. 368, 620-630 (1996).</li><li>Cantos, R., Cole, L. K., Acampora, D., Simeone, A., 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=Patterning+of+the+mammalian+cochlea.">Patterning of the mammalian cochlea.</a> Proc Natl Acad Sci U S A. 97, 11707-11713 (2000).</li><li>Whitfield, T. 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 authors have nothing to disclose.

The here described method of in ovo micro-electroporation is optimized for gene transfer into the developing auditory organ. It is compatible with plasmid DNA-based expression vectors typically used to manipulate gene function/expression. The timing of electroporation at E4 is optimal for investigations that focus on HC development in the auditory organ. The most critical steps are micro-injecting the DNA into the otic vesicle lumen without going too deep with the needle and damaging the otic vesicle (see Fig. 1A-D), targeting the DNA into the anterior-ventral domain of the otic vesicle by placing the electrodes accordingly (see Fig. 1C), and delivering the optimized electroporation parameters of 4 pulses at 12 V. These are optimized electroporation parameters and optimized concentration of plasmid DNA (~3.5 -&#160;4 &#181;g/&#181;l) for specifically targeting the presumptive auditory organ at E4. Initially we started out conducting electroporations at 6 V, which yielded no fluorescent signals. The voltage was incrementally increased by using 8 V&#160;and 10 V, with 12 V&#160;being optimal. Similarly, the number of pulses to deliver and the pulse durations and spacing of pulses were optimized. At 12 V, 4 pulses with 100 msec duration and 200 msec spacing is ideal. These parameters are not only ideal for obtaining efficient gene transfer at E4, but are also ideal for an embryo survival rate of approximately ~50%. If no fluorescent signal is obtained within 24 hr&#160;of electroporation, the following should be considered: 1.) Make sure that the plasmid DNA concentration is at least 3.5 &#181;g/&#181;l. 2.) Adjust the electroporation parameter by 2 V, for example to 10 or 14 V. As to obtaining E4 (HH24-25) stage embryos within 117 hr&#160;of incubation time (see Fig. 1D), the incubator temperature for the eggs should be optimized between 37 - 39 &#176;C with more or fewer hours of incubation time needed than 117 hr&#160;depending on the temperature used, because there are variations in holding and running temperatures using different incubators among different laboratories. Various thymidine analogs including BrdU (5-Bromo-2&#8242;-deoxyuridine) or EdU can be used to study in ovo cell proliferation. However, the use of either compound has to be optimized for each developmental stage as too much of the compound proves toxic for cell/embryo viability and too little of the compound proves ineffective. This method describes the optimized use of EdU at a concentration of 0.25 mg/ml applied as 50 &#956;l onto the whole embryo in ovo specifically for studying cell proliferation and cell-cycle-exit from E4 onward.
In ovo electroporation as a method of gene manipulation has some limitations. A relative large number of eggs are required for electroporations at E4 with appropriate control constructs and for the experimental conditions. This is due to a proportion of eggs, approximately ~27%, being unusable for electroporation due to a combination of lack of fertilization, developmental defects, and embryonic death. Another important factor to consider is survival rate of the embryos after electroporation. The survival rate is approximately ~50 %. This is due to the fact that older embryos at E4 do not survive well after being manipulated; younger embryos are more resilient and tolerate being manipulated better. Expressions of the genes of interest after electroporation at E4 are focal and localized throughout the entire cochlear duct including non-sensory and sensory tissue as well as the auditory ganglion. The reason for the more focal and localized expression is because the target of the electroporation is the presumptive, undeveloped auditory organ at E4, before the chicken auditory organ starts to grow out at E5 and elongates into its characteristic sickle-shaped form by ~E9 1. Lastly, the method is transient where expression signals only remain for ~4 days, because the genes of interest used here do not integrate into the genome. However, the method can be used with appropriate plasmid-based expression constructs&#160;that do have the ability to integrate into the genome.
Mastering the electroporation method requires some practice, trained eyes and hands, and expertise in working with chicken embryos. The references provided for inner ear development and chicken developmental atlases serve as good guides and are good starting points for a novice. After mastering the method with these optimized electroporation parameters, the method can be used for manipulations in the closed otic vesicle from E2 to ~E4.5. Although for electroporating at E2 and E3, 10 V&#160;should be used instead of 12 V. For electroporation at E1 targeting the otic placode and otic cup, customized platinum electrodes with modified electroporation parameters can be used 10. The earlier manipulation can be used to study earlier events in otic development including inner ear neurogenesis and the development of vestibular sensory organs 8-10. In addition to studying the development of the auditory organ this method also lends itself to study the development of the innervating auditory ganglion. Analysis of GFP expression in electroporated specimens revealed that targeting specifically the anterior-ventral otic vesicle not only targets the sensory domain within the neural-sensory competent (NSD) epithelium but also targets the neuronal population that reside within the NSD. Afferent neurons innervating the vestibular and auditory sensory organs are primarily of otic placodal origin. Neurogenesis occurs over an extended time period approximately from ~E1.5 -&#160;E4.5 in the chick, where neuroblasts, undergoing intense cell proliferation continuously delaminate from the NSD epithelium of the otic cup (~E1.5) and otic vesicle (~E2.5 -&#160;E4.5) and populate as differentiating neurons the cochlear-vestibular ganglion (CVG; VIII cranial nerve) 8,10,13,22.
Moreover, with some modifications vestibular organs can be targeted with the method presented. For example, the presumptive anterior crista can be targeted at E3, which lies on the dorsal anterior tip of the anterior-ventral NSD 9. Electroporating at E4 targeting the anterior-ventral otic vesicle yields focal yet robust GFP transcript expressions within sensory and non-sensory tissue in all of the sensory vestibular organs, namely the cristae, and utricluar and saccular maculae (data not shown).
In conclusion, the in ovo method presented here is specific for conducting gain-and loss-of functions starting at E4 and for studying proliferation and differentiation in the developing chicken auditory organ. Moreover, this method can be easily adapted for studying other inner ear developmental phenomena, including inner ear neurogenesis and the development of vestibular organs.

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

<table border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green FCF Powder</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EdU Powder</td>
			<td>Invitrogen</td>
			<td>E10187</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Click-IT EdU Alexa Fluor 555 Imaging Kit</td>
			<td>Invitrogen</td>
			<td>C10338</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HiSpeed Plasmid Midi Kit (25)</td>
			<td>Qiagen</td>
			<td align="right">12643</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECM 830 ElectroSquarePorator&#160;</td>
			<td>BTX Harvard Apparatus</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td>45-0216</td>
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			<td>World Precision Instruments Inc.&#160;</td>
			<td>M3301R &#38; M3301L</td>
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			<td>World Precision Instruments Inc.&#160;</td>
			<td>M10</td>
		</tr>
		<tr height="21">
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			<td align="right">5479</td>
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			<td>World Precision Instruments Inc.&#160;</td>
			<td align="right">14120</td>
		</tr>
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			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
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		<tr height="21">
			<td height="21" style="height:21px;">2.5x2.5 mm Box Platinum Heating Filament</td>
			<td>Sutter Instrument Co.</td>
			<td></td>
		</tr>
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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>Fisher Scientific</td>
			<td>O122-1</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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		<tr height="21">
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			<td>Becton Dickinson and Company (BD)</td>
			<td align="right">309657</td>
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			<td height="21" style="height:21px;">Disposable 21 Gauge Needles</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td align="right">305122</td>
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			<td>CUY611P3-2</td>
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			<td>CUY580</td>
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			<td>23-400-101</td>
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	</tbody>
</table>

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 the anterior-ventral aspect of the otocyst, that gives rise to the BP (Figure 1 E-E&#39;; white arrow) and GFP expression can be followed over a time course of up to approximately ~4 days (Figure 1 E-H). RNA in situ hybridization (ISH) was used to determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. To determine whether GFP was taken up by auditory sensory progenitors, ISH experiments were performed on adjacent BP sections. Within the developing BP sensory progenitors were identified by their expression of Sox2 (Figure 1 K and N) and Lunatic fringe (Lfng) (Figure 1 J). These experiments demonstrate that sensory progenitors throughout the developing BP, as shown for the base (Figure 1 I; black bracket) and apex (Figure 1 L; black bracket), were successfully targeted and express GFP after 68 hr of electroporation (Figure&#160;1 I and L; black brackets). In addition, GFP was expressed outside the sensory domain in non-sensory epithelial cells (Figure 1 I and L; red arrows) and in the auditory ganglion (Figure 1I and L; ag, yellow arrows) marked by Neurod1 expression (Figure 1M). In a typical experiment GFP expression in targeted cells persisted for up to ~4 days (Figure 1 H); this allows the investigator to analyze the spatial and temporal pattern of sensory progenitor cell-cycle withdrawal and differentiation in the developing BP. To determine the proliferative behavior of sensory progenitors, the&#160;thymidine analog EdU&#160;was added at the time of electroporation at E4. Four&#160;days later, EdU incorporation in the developing BP was analyzed in tissue sections using &#34;click&#34; chemistry. Immuno-labeling for the HC-specific protein MyosinVIIa (Myo7a) was used to identify differentiating HCs in the developing BP. In the apical portion of the BP Myo7a+ HCs were frequently EdU+ (Figure 1 P-P&#39;; P&#39;&#39;, white arrows), whereas in the base only few Myo7a+ HCs had incorporated EdU (Figure 1 O-O&#39;; O&#39;&#39;, white arrows), suggesting that at the time of EdU addition at E4, sensory progenitors in the base are already largely post mitotic. These results confirm previous studies of opposing gradients of cell-cycle exit and HC differentiation in the chick BP 6,7.
<img alt="Figure 1" src="/files/ftp_upload/53864/53864fig1.jpg" /> 
Figure 1.&#160;In Ovo Electroporation &#38; In Ovo Cell Proliferation Assay.&#160;(A-D) Brightfield images of electroporation method. In ovo micro-injection into the right otic vesicle (B) and placement of electrodes after micro-injection targeting the anterior-ventral area in the otic vesicle (C). Embryo at stage HH24-25 after micro-injection and electroporation (D; green otic vesicle, black arrow). (E-H) Green fluorescent protein (GFP) expression after electroporation. (E) Brightfield image and GFP expression in the otic vesicle (outlined in red) at 18 hr. (E&#39;) GFP expression at 18 hr&#160;(otic vesicle, white arrow). (F-H) White arrows point to GFP expression at 40 hr&#160;(F, otic vesicle), 68 hr&#160;(G, BP), and at 88 hr&#160;(H, BP). (I-N) Adjacent cochlear tissue-sections of the BP probed for GFP, Lfng,Sox2, and Neurod1 transcripts by RNA in situ hybridization at 68 hr. Lfng (J, black bracket) and Sox2 (K and N, black bracket) mark the sensory epithelium and Neurod1 (M) marks the auditory ganglion (ag). GFP is expressed within sensory (I and L, black bracket) and non-sensory tissue (I and L, red arrows), and the auditory ganglion (I and L, yellow arrows). (O-P&#39;&#39;) Fluorescent images of MyosinVIIa (Myo7a)/EdU labeling. (O&#39;-O&#39;&#39; and P&#39;-P&#39;&#39;) Higher magnifications of O and P. (O&#39; and P&#39;) More EdU labeling is present within the sensory epithelium at the apex (P&#39;, white bracket) than at the base (O&#39;, white bracket). O&#39;&#39; and P&#39;&#39;) Merged fluorescent images, white arrows point to Myo7a(+)/EdU(+) hair cells at the apex (P&#39;&#39;) and the base (O&#39;&#39;). More EdU(+) cells are present within the auditory ganglion at the apex (P, ag) than at the base (O, ag). (sac; saccule in O). (A, Anterior and V, Ventral in panels B and E). Scale bars 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53864/53864fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

gene-transfer-into-chicken-auditory-organ-ovo-micro

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.4K Views.  The Johns Hopkins University, School of Medicine. 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. 

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

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 ...

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, ...

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. ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

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 ...