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">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Ciprofloxacin Hydrochloride</td>
			<td dir="LTR" style="width:192px;">Cellgro</td>
			<td dir="LTR" style="width:161px;">61-277-RF</td>
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			<td dir="LTR" height="43" style="height:43px;width:316px;">Glass Dish 60mm</td>
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			<td dir="LTR" style="width:161px;">23062-6015/23064-6015</td>
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			<td dir="LTR">Kimble Chase</td>
			<td style="width:161px;">23064-10015/23062-10015</td>
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			<td dir="LTR" style="width:161px;">11251-10</td>
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			<td dir="LTR" style="width:161px;">P35G-0-10-C</td>
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			<td dir="LTR" style="width:161px;">356237</td>
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			<td height="43" style="height:43px;width:316px;">Culture Dish, 60 X 15 mm</td>
			<td dir="LTR" style="width:192px;">Becton Dickinson</td>
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			<td height="43" style="height:43px;width:316px;">Tissue&#160; Culture Dishes</td>
			<td dir="LTR" style="width:192px;">Greiner Bio-one</td>
			<td style="width:161px;">639160</td>
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			<td dir="LTR" height="43" style="height:43px;width:316px;">Phosphate Buffered Saline</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">10010-023</td>
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			<td dir="LTR" style="width:161px;">4021768</td>
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			<td dir="LTR" height="43" style="height:43px;width:316px;">Fluoromount</td>
			<td dir="LTR" style="width:192px;">Southern Biotech</td>
			<td dir="LTR" style="width:161px;">0100-01</td>
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			<td height="43" style="height:43px;width:316px;">Conical Tubes, 15ml</td>
			<td style="width:192px;">Greiner Bio-one</td>
			<td style="width:161px;">188261</td>
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			<td height="43" style="height:43px;width:316px;">Myosin 6</td>
			<td>Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6791</td>
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			<td height="43" style="height:43px;width:316px;">Myosin 7a</td>
			<td style="width:192px;">Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6790</td>
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			<td height="43" style="height:43px;width:316px;">TuJ1</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:161px;">T2200</td>
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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. 

视频: Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by 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.

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

鸡胚全视网膜外植体电穿孔和化学试剂处理

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

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.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

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

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

小鼠胚胎肾的解剖与培养

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

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.

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

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

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...