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.

注：使用活的动物所有协议必须审查和批准的机构动物护理和使用委员会（IACUC），并且必须遵循正式批准为实验动物的护理和使用方法。所有的解剖应该使用无菌技术在洁净层流工作台进行。手套和口罩，如果需要的话，应在此过程中磨损。 1.解剖胚胎小鼠耳蜗<ol><li>设置为尔蒂植文化的器官： <ol><li>通过接通UV光约30分钟灭菌的层流组织培养罩并消毒以在使用前70％的乙醇的表面。此外，灭菌的所有解剖仪器，包括精细清扫工具和的Sylgard涂覆菜肴用于经由高压釜胚胎解剖或通过浸泡在70％乙醇中在使用前至少20分钟。倒掉70％乙醇使菜肴和仪器空气干燥洁净层流CLEAñ板凳后才能使用。 </li><li>制备无菌Hank氏平衡盐溶液（HBSS）/（4-（2-羟乙基）-1-哌嗪乙磺酸）（HEPES）加入10倍的HBSS和0.5％的HEPES 10％，将pH调节至约7.2并过滤消毒混合物最终溶液并储存在4℃。开展冷冻HBSS / HEPES解决方案的所有解剖整个过程中，以更好地保护组织。 </li><li>与基底膜基质加入5毫升冷（4℃）的无菌的Dulbecco改良的Eagle培养基（DMEM）中于基底膜基质的300微升等分试样在无菌15mL的聚丙烯管外套的玻璃底菜肴。通过涡旋的内容混合，并添加150微升基底膜基质的DMEM混合物中的每个培养皿的中心上，使得它覆盖以及存在于该盘的中心的整个培养。覆盖菜肴和商店在37℃培养箱使用前至少45分钟。 </li><li>倒入4-5毫升冷冻HBSS到日稀土无菌聚苯乙烯培养皿中，并在引擎盖假期关闭。 </li><li>通过加入9毫升DMEM中，加入100μlN2补充剂，10μg/ ml的环丙沙星和1ml的FBS的制备培养基在无菌15mL的聚丙烯管中。 </li></ol></li><li>隔离胚胎： 注：在此过程中从小鼠胚胎培养耳蜗外植体，收获内耳从胚胎天（E）13颞骨受精后的一天，被视为E1 9。该协议也可以用于与胚胎小鼠内耳开始从E12到E18。 <ol><li>安乐死定时怀孕的鼠标采用CO 2和颈椎脱位或适当批准的协议牺牲的动物。开展安乐死远离层流组织培养罩，以保持无菌的组织培养室。 </li><li>放置安乐死鼠标上纸巾朝上腹部和消毒腹部用70％乙醇。 </li><li>通过抓住皮肤打开腹腔用弯钳，一手抓削减表皮和肌肉以及用另一只手的剪刀中线。使两侧两个垂直切割并小心地拔出子宫提起了双侧子宫角钳和结缔组织组织使用剪刀下面分离。 </li><li>将胚胎链中含有冷冻夹层溶液（HBSS / HEPES混合物），并转移到层流洁净工作台培养皿。 </li><li>从胎盘中小心取出胚胎，将它们放入含夹层解决方案的无菌培养皿之一。如果溶液变成血腥，将它们移动到一个新的培养皿中。 注意：此时，胚胎可以使用Theiler分期引导上演。 </li><li>通过在颈部搭配一双干净的镊子或剪刀捏杀头的胚胎，然后将头含冷冻术解决方案一个新的培养皿中。 </li></ol></li><li>夹层的内耳： <ol><li>代替邻NE胚胎头含冷解剖无菌SYLGARD菜</li><li>解决方案。在解剖显微镜下工作在〜1.6X的倍率，通过将Minutien销周围或接近眼睛区域（ 图1A）固定的胚胎的头。 </li><li>使用两对无菌镊子小心取出皮肤和开放的头骨上沿中线（ 图1B）背侧。从颅腔和位于颞骨内内耳可以在此阶段（ 图1C，D） ​​被识别除去大脑。的血管，通常是围绕内耳本的衬里 ​​，可以用作一个里程碑用于识别内耳在此阶段（ 图1D）。 </li><li>通过将镊子组织下面，隔离颅骨和转移基地内耳含冷解剖解决方案的新菜剖析从颞骨内耳（ 图1E，F）。 </li></ol></li></ol> 2，代尔蒂植文化机关注：在这个阶段的发展，组织是软骨，可以使用镊子容易解剖。 <ol><li>东方使得腹侧朝上（ 图2A）和通过内耳（ 图2B）的前庭部轻轻插入的Minutien销的尖端稳定内耳内耳。 </li><li>使用超细钳子切断通过使用近卵圆窗钳子的一端，并小心地取出从耳蜗（ 图2C）的软骨的切口打开覆软骨。 注：以确保钳不插入过深地进入软骨作为覆软骨有时融合耳蜗管在这种情况下是有帮助的使用封闭镊子从底层耳蜗管释放软骨通过刮擦下面这一点很重要surface软骨。在这个阶段的发展，耳蜗螺旋只有四分之三的长度一转。 </li><li>接着，露出的感觉上皮通过将钳子在耳蜗管的优选区域，无论是在碱或在最顶点，并轻轻拉出耳蜗（ 图2D）的屋顶。 注：耳蜗的屋顶必须完全去除，因为它可掩盖感觉上皮阻碍分析。 注：此过程必须做轻轻的，因为它是易撕感觉上皮。 </li><li>作为最后一步，小心地从露出感觉上皮去除下层的结缔组织，使得耳蜗植体的碱是均匀平坦的。 </li><li>从使用镊子内耳前庭部隔离解剖耳蜗。这可以步之前或之后2.4（ 图2E）中进行。 </li><li>转让解剖耳蜗感觉上皮到地下室membraNE基质包被的培养使用1.5毫米无菌的压（ 图2F）好。 </li><li>东方耳蜗植与上皮的腔表面朝上，并吸出基底膜基质的DMEM溶液小心弄平组织并轻轻加入到培养皿它与150微升新鲜培养基更换。应注意，以确保每个外植体很好地粘附涂覆的玻璃盖玻片，并且不漂浮在培养基中。确保镊子尖上仰，同时粘贴到玻璃，以避免损坏耳蜗外植体。 </li><li>轻轻在37℃用5％CO 2的培养皿转移到无菌150毫米培养皿并将其放置在组织培养培养箱中的时间所需的时间，通常为3-6 的体外天（DIV）。经过1 DIV，检查解剖显微镜下的文化，以确保耳蜗外植体已经很好地结合到培养皿。 </li><li>后cubation 体外的时间所需长度，培养物处理用于免疫组织化学。 注：外植体培养通常孵育6 DIV相当于发育阶段，P0。孵育体外后，培养物可以用于进一步的下游应用，如免疫组织化学（ 图3B）进行处理，RT-PCR， 原位杂交，和/或免疫印迹。 </li></ol> 3.电穿孔介导的基因转移<ol><li>设置的电穿孔： <ol><li>消毒1100毫米SYLGARD被覆玻璃皿通过高压灭菌或浸泡在70％乙醇中约20分钟，并允许空气干燥在使用前净化台。 </li><li>从选择使用合适的马克西或MIDI-准备套件的表达载体制备DNA。在这个协议中使用的表达载体的pIRES2-Atoh1.EGFP和pCLIG-NeuroD1.EGFP。 DNA的终浓度应至少为1微克/微升无菌的DNA /无RNA酶的水。 </li></ol></li><li> DNA电穿孔进入胚胎耳蜗植： <ol><li>加10微升质粒DNA溶液至新鲜百毫米的Sylgard涂覆培养皿并传送一个完整解剖耳蜗植（来自步骤2.5获得）到DNA溶液中。 </li><li>随着朝上上皮腔面，倾斜耳蜗稍微因此，它是垂直于盘的平面。 </li><li>将电极极板与负桨（阴极）向感觉上皮和位于邻近所述耳蜗的底部的正桨（阳极）的耳蜗的任一侧。 注意：由于DNA被带负电的，它从负到正电极和通过将相邻的感觉上皮探头的阴极端迁移;该DNA将主要经历的感觉上皮的表面上。 </li><li>通过电穿孔，提供24 mV的9至10个脉冲，30毫秒脉冲持续时间与T上的脚踏开关他电穿孔，然后添加100微升暖培养基至电穿孔耳蜗。 </li><li>对于所有的耳蜗植和所有感兴趣的基因的DNA的重复步骤3.2.1-4和电穿孔耳蜗转移到基底膜基质被覆培养皿电镀（步骤2.7）。培养都在37℃，5％CO 2的湿润培养箱的耳蜗电的时间所需长度将随后进行免疫细胞化学处理。 注：从CD1小鼠（约8-12幼仔），整个垃圾耳蜗可以分离，显微，电和镀&lt;4小时。 </li></ol></li></ol> 4.分析耳蜗外植体文化注：该培养通常孵育6 DIV相当于发育阶段，P0。孵育体外后，将培养物固定并处理用于免疫细胞化学。 <ol><li> INC所需的长度后，ubation，除去培养基，并迅速冲洗在磷酸盐缓冲盐水（PBS）的耳蜗植和15分钟，在室温下孵育在4％多聚甲醛（在PBS中制备的）。 </li><li>通过加入PBS中持续15分钟，洗固定液和冲洗在PBS三次。 </li><li>透用PBS-T（PBS + 0.5％吐温）中30分钟，阻断用含有10％山羊血清的PBS-T之前。 </li><li>孵育在一次抗体溶液含有在PBS-T中的第一抗体，用1％山羊血清过夜，在4℃。 </li><li>用PBS-T的每个15分钟加入适量的Alexa标记的二抗前，在室温下洗耳蜗三次。 </li><li>由于耳蜗植是在玻璃盖玻片培养可以通过从培养皿中分离盖玻片和安装在滑动单独处理。这可以通过在室温下浸泡在培养皿中OS30溶剂为至少1小时来实现。使用无菌刀片，从D脱离盖玻片十岁上下，并安装在一个幻灯片，并在荧光显微镜下可视化。 </li></ol>

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No conflicts of interest declared.

所有的小鼠内耳的膜迷路，包括内细胞的感官，非感觉和螺旋神经节神经元均从placodally衍生otocyst相邻外胚层的后脑，围绕E8 10-14而得。在E11中，otocyst的腹侧区域延伸，以形成所述蜗管并作为发展下去，一组中的耳蜗内的上皮细胞，以及在otocyst的其他区域，成为指定为prosensory补丁，这将随后产生不同类型的mechanosensory毛细胞和非感觉支持细胞。在显影耳蜗内毛细胞中的一个行和三行外毛?...

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

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">HBSS</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:161px;">14065-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">HEPES</td>
			<td>Gibco</td>
			<td style="width:161px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Dulbecco&#8217;s Modified Medium</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">12430-054</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Fetal Bovine Serum</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">10082</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">N-2 Supplement (100X)</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
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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.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

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

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

Research

JoVE Journal

Developmental Biology

12.8K 次观看.  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. 