我们感谢多丽丝·K.吴博士为表达质粒和原位探测，约翰·霍普金斯大学中心的感官生物成像设备和中心的听力和平衡。这项工作是由NIDCD格兰特T32 DC000023到LE支持

鸡蛋和未孵化的胚胎关心和道德和人道待遇。对于未孵化的胚胎使用的所有协议是由动物护理和使用委员会在医药，马里兰州巴尔的摩的约翰霍普金斯大学医学院的批准。 1.鸡蛋和表达结构的制备<ol><li> 蛋: <ol><li>孵育3 - 4打受精鸡蛋（ 鸡内金 ）趴在其两侧平在37℃。 <ol><li>后培养24小时，拉3立方厘米蛋白与来自蛋的圆形后端的注​​射器和密封用透明胶带的孔中。这产生胚胎和蛋壳之间的空气室，从而允许易于接近胚胎无胚胎切割的窗口上的蛋19的顶部检修时粘到外壳的。 </li><li>在孵化时间117小时，按照汉堡和汉密尔顿20发育阶段阶段胚胎在E4（HH24-25）（参见图1D）。 </li></ol></li></ol></li><li> 表达构造: <ol><li>准备使用试剂的质粒DNA并用市售制剂试剂盒的制造商提供的协议。在制备的最后步骤，洗脱DNA进500微升TE液（Tris-EDTA）缓冲设置在试剂盒中到1.5毫升管。 </li><li>乙醇沉淀通过加入3M乙酸钠的50微升和1ml 100％乙醇的到管集中它的DNA。放置管在-20℃CO / N。 </li><li>离心反应管30分钟，在4℃下14,000rpm下。除去上清液并空气干燥沉淀在室温10分钟。溶解在15微升TE缓冲液将沉淀，这应该产生〜4微克/微升的浓度。 注:此处使用的对照表达构建体是机动设备-IRES-GFP的 10，它是由一个鸡β肌动蛋白启动子驱动，或PCI-H2B-IRES-RFP <s向上&gt; 10，这是由CMV启动子驱动。 </li></ol></li></ol> 2.鸡卵类微型电穿孔和在卵细胞增殖试验<ol><li> 在表达构建和微电的大毛微注射: <ol><li>使用微吸管拉出器与一个2.5×2.5 2mm的盒铂加热灯丝下面优化参数拉玻璃毛细管成细针状:压力= 500，分组= 600℃，拉= 64，速度= 105，和时间= 150毫秒（ 见表1）。 </li><li>成立了左和右手微操纵阶段侧翼显微镜（参见图1A）。右手微型机器人拥有玻璃毛细管针和左手微机器人保持电极（ 图1A）。 </li><li>准备拉细玻璃毛细管针显微注射切割而在显微镜下工作的针用钳子的前端。 </li><li>使用微机械手与质粒DNA 2.5微升含0.1％固绿着色显微镜下工作时用手手动填充针。 </li><li>放置蛋一个模具/蛋保持装置内侧躺着（ 见图1A）。切使用剪刀19（ 图1A）的鸡蛋顶端圆窗。 </li><li>而在显微镜下工作，小心地打开两个膜覆盖使用镊子胚胎。 </li><li>而在显微镜下工作时，通过微注射递送约〜0.5微升质粒DNA到右侧耳泡腔用右手通过使用显微操作和通过微电手动。 <ol><li>这样做，而在同一时间使用左手牵着与用右手的微操纵器（ 图1B-D）的DNA微注射右耳泡腔GA钳子稳住胚胎的头，因为胚胎的头逢低在舞台E4。不要微以往注入耳泡腔，因为这会损害耳泡。 </li><li>微注射后立即，同时在显微镜下工作，可以使用左手的微机械手放置的正极（阳极）2毫米的铂金电极前腹向右耳泡和并联负2毫米电极（阴极）相距1毫米（ 见图1 C）。提供4个脉冲在12 V与100毫秒的持续时间和200毫秒间隔。左耳泡用作内部未处理的对照。 注:在1X PBS蘸棉签电穿孔的清洁电极。 </li></ol></li></ol></li><li> OVO中的细胞增殖试验: <ol><li>立即电穿孔后加入50微升的0.25毫克/毫升的EdU在1×PBS中由50微升的EdU溶液手动放置到全胚胎卵内使用移液管。密封该鸡蛋用胶带，并返回到培养箱中18 - 96小时。 </li><li>小心地取下胶带并检查后，18耳囊中荧光GFP或RFP信号胚胎-使用标准的荧光显微镜24小时（ 见图1 EE&#39;）。如果荧光信号存在 ​​，在这一点上收获胚胎分析或用胶带重新密封，并将其返回到培养箱中收获和在稍后阶段分析（ 见图1 FP&#39;&#39;）。 注: -电10 7小时后GFP信号与机动设备-IRES-GFP表达构造6是显而易见的。 </li></ol></li></ol> 3.胚胎收获和组织处理的RNA原位杂交和免疫组化<ol><li>收获使用镊子的胚胎。冲洗冷1X PBS胚胎。取出胚胎的头，通过它的脖子割头使用镊子，然后将头到4％多聚甲醛O / N在4℃。使用钳子以允许头部内的4％多聚甲醛的渗透穿刺大脑。 </li><li>脱水30％蔗糖的头（在1X PBS）O / N在4℃。 </li><li>通过快速冷冻在干冰和甲基丁烷（2-甲基丁烷）的浆料在安装中冷冻保护头。另外，快速冻结元首在用液态氮冷冻保护网上平台。在-80℃保存安装头。 </li><li>冷冻切片头到12微米厚的组织切片，并收集所有的内部含有耳组织切片上superfrosted显微镜载玻片上。 </li><li>执行原位杂交和免疫组织化学标准RNA如前所述4,21，和分析的EdU细胞增殖以下由该试剂盒的生产商提供的协议。 注:毛细胞可以用多克隆鉴定α-MyosinVIIa（MYO7A）的tibody（1:1000，变形杆菌Biosciences公司）和荧光标记的第二抗体（1:250，山羊抗兔AlexaFluor 488; Invitrogen）中。 </li></ol> 4.图像捕捉和处理<ol><li>采取与数字影像设备的图像。图像免疫结果与标准荧光显微镜和使用标准宽视场显微镜（从5倍到40倍的放大倍率） 的原位的结果。 </li><li>处理使用图像处理软件的图像。 </li></ol>

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The authors have nothing to disclose.

的卵内的微电穿孔的这里描述的方法是用于基因转移到显影听觉器官优化。它与典型地用于操纵基因功能/表达质粒DNA为基础的表达载体相容。电在E4的时机是最佳的，专注于HC发展的听觉器官调查。最关键的步骤是微型喷射的DNA导入耳泡腔不打算与针太深并损坏耳泡（参照图1A-D）中 ，靶向DNA插入通过耳囊的前腹域相应地放置电极（参照图1C），并在12 V...

尽管在形态和哺乳动物和鸟类听觉感觉器官之间的差异的蜂窝图案，分子因素和负责感官HC发展途径被认为是进化上保守的1-3。基底乳头，里面听觉碳氢化合物及其周围旺，发展为内耳听囊的outpocketing。早期HC和SC祖细胞的池耳placode /耳杯的神经感官能力的域（NSD）内指定。命运映射数据提供证据表明，神经元和感官谱系被链接，并从设在耳杯的前-腹侧区域或在小鼠和鸡4,5-听囊的NSD出现。首先，成神经细胞从NSD脱层到产生听觉前庭神经节，最终分裂成听觉和前庭神经节的神经元。这些神经元支配内耳和原子核在脑干的感觉碳氢化合物。细胞届在保持在NSD被认为会引起各种感觉补丁，包括听觉感觉器官，由机械性传感感官碳氢化合物及其相关的SC的。 在鸡和鼠两个，听觉器官感觉祖细胞周期退出和HC分化发生在相反的梯度。在小鸡，听觉祖细胞周期出口开始围绕〜E5和从基部到顶点的进行，并且从中心到周边6。一天后，HC分化开始在顶点和前进到基座7。在鸡研究内耳的发展提供了许多技术优点的功能机制可以在相对 ​​于在子宫内的其他模型系统，这需要复杂的手术操纵胚卵内相对容易进行调查。这里所描述的方法使用在卵内的微电靶向感兴趣的基因中的推定basila- [R乳头区前，在腹侧在专门的E4耳泡。 电穿孔卵内是很好地建立和常用8-14的技术。电穿孔技术的原理是基于这样的事实，核苷酸（ 例如，质粒DNA，合成DNA或RNA寡核苷酸）被带负电荷。将DNA注射到感兴趣的组织。当电流被置于穿过组织，目前在细胞壁打开瞬态孔，并允许该DNA的摄取，因为带负电荷的DNA的朝正电极（阳极）流动。对于增益的功能的实验中，感兴趣的基因通常亚克隆到包含绿色荧光蛋白（GFP）或红色荧光蛋白（RFP）适当的表达盒的表达质粒。对于失功能实验基于质粒的显性负阻抑构建体，或RNAi，或吗啉代构建体是Commonly使用15,16。以抑制微小RNA（miRNA）的函数的miRNA海绵结构，这是典型的质粒基于，可以使用17。在这里描述的方法允许调查控制细胞增殖和分化的听觉器官的分子机制，因为卵内的微电穿孔法可以很容易地与卵内细胞增殖试验加入的EdU整个胚胎相结合。 在电穿孔和另外埃杜是在E4在耳泡，这是细胞周期退出的听觉器官发病的前一天进行。听觉器官通常进行18分析 - （HC分化过程中）96小时电穿孔后在E5（感官祖细胞周期出口的发病），E6（HC分化的开始），和E7;后来高达〜E9（HC分化的终点）。这种基因转移的电穿孔法是短暂持续大约〜4天，becau本身的兴趣的基因不整合到基因组中，但该方法适用于具有适当的质粒DNA为基础的表达载体，其是有整合到基因组中，如TOL2介导的基因转移11的能力使用。用这种方法强大的GFP或RFP表达持续〜4个天耳蜗，在此之后的信号褪色，但提供了充足的时间来研究听觉器官的复杂发展。这在卵基因转移方法新颖，允许专门针对在E4假定的听觉器官，这是最佳的，专注于HC发展的听觉器官调查。这是一个很好的补充替代方法，这在电穿孔更加年轻的发育阶段的11,12或相比，使用体外基底乳头外植体培养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">
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			<td>Invitrogen</td>
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			<td>45-0216</td>
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			<td>World Precision Instruments Inc.&#160;</td>
			<td align="right">5479</td>
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			<td height="21" style="height:21px;">Scissors for Eggs 12 cm long curved, 12 mm extrafine blades; Spring Scissors</td>
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			<td align="right">14120</td>
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			<td height="21" style="height:21px;">Micropippette Puller for pulling needles</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
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			<td height="21" style="height:21px;">2.5x2.5 mm Box Platinum Heating Filament</td>
			<td>Sutter Instrument Co.</td>
			<td></td>
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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>
		</tr>
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			<td height="21" style="height:21px;">Hamilton Glass Syringe 100 &#956;l</td>
			<td>Hamilton</td>
			<td>80601 Model 710LT</td>
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			<td height="21" style="height:21px;">Mineral Oil (Heavy) for Hamilton Glass Syringe</td>
			<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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			<td>Becton Dickinson and Company (BD)</td>
			<td align="right">309657</td>
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			<td>Bulldog Bio. / Nepagene</td>
			<td>CUY611P3-2</td>
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			<td>Bulldog Bio. / Nepagene</td>
			<td>CUY580</td>
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			<td>Fisher Scientific</td>
			<td>23-400-101</td>
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	</tbody>
</table>

gene transfer into the chicken auditory organ by in ovo micro-electroporation

鸡胚胎是研究胚胎发育为基因功能的操作可以与卵内相对容易地进行理想模型系统。内耳听觉感觉器官是我们听到的能力至关重要。这房子是由机械力转导毛细胞（HCS）一个高度专业化的感觉上皮和周围神经胶质样支持细胞（SCS）。尽管在听觉器官的结构差异，调节听觉器官的发展的分子机制的哺乳动物和禽类动物之间是进化保守的。在卵内电穿孔在很大程度上限制为E1的早期阶段- E3。由于在E5听觉器官的相对发展较晚， 在OVO电过去E3听觉器官的操作由于鸡胚在稍后阶段先进的开发都很难。这里介绍的方法是一种用于在靶向的目的基因瞬时基因转移方法阶段E4 -通过卵内的微电E4.5在显影鸡听觉感官。这种方法适用于GAIN-和失功能与常规质粒基于DNA的表达载体，可与卵细胞增殖试验通过在加入的EdU（5-乙炔基-2&#39;-脱氧尿苷）的全胚胎被组合电的时间。使用绿色或红色荧光蛋白（GFP或RFP）表达质粒可以让实验者快速确定是否电针对成功发展内耳听觉部分。在该方法中纸张，GFP电穿孔样品的代表性实例示出;胚胎收获18 -电后96小时和听觉器官的亲感觉区GFP的目标是通过RNA原位杂交证实。该方法本文还提供了使用胸苷的优化协议模拟的EdU分析细胞prolif关合作;提供结合免疫标记，然后点击的EdU化学基于的EdU细胞增殖试验的一个例子。

在这个自由绿色荧光蛋白的方法纸张质粒DNA（GFP）的表达盒被定位到显影鸡基底乳头（BP）与与100毫秒脉冲持续时间12 V和4个脉冲和200毫秒的间隔进行优化参数，产生〜 50％的胚胎成活率和质粒DNA靶向到BP的效率。在胚胎发育天然GFP表达的荧光成像显示电穿孔的此方法优选地定位到听囊的前腹方面中，产生该BP（ 图1 EE&#39;;白色箭头）和GFP表达可以遵循以上的时?...

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

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

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

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

Research

JoVE Journal

Developmental Biology

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

9.4K 次观看.  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. 

视频: 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.

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 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.

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 ...

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.

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

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.

珠嫁接到发展鸡胚四肢识别信号转导途径影响基因表达

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).

一本小说<em&gt;防爆大毛</em&gt;带技术来改变心内血流动力学在鸡胚胎系统

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.

在奥运电击鸡听觉脑干中

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.

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 ...

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.

三维脊髓前庭和听觉感官器官的培养

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.

使用Trowell型器官培养研究牙科干细胞的调节

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.

分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

基因转移到鸡听觉器官通过

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