我们要感谢Drs。 Leslayann Schecterson，Yuan Wang，Andres Barria和Ximena Optiz-Araya太太，为协议设置和提供质粒提供初步帮助。这项工作得到NIH / NIDCD授权DC013841（JTS）的支持。

所有程序均经西北大学机构动物护理和使用委员会批准，并按照国家卫生研究院"实验动物护理和使用指南"进行。 蛋处理<ol><li>从当地的供应商处购买受精卵。在孵育前不要超过5天，将冰箱中的鸡蛋在13°C储存。 1周后胚胎活力明显下降。 </li><li>用70％乙醇清洗每个鸡蛋，然后放入培养箱中。 </li><li>在温度设定在38°C〜50％湿度的孵化器中，在其侧面设置1-2打鸡蛋。这确保了电穿孔的适当胚胎定位和最佳的生存能力。 注意:孵化48-52小时后，鸡蛋将在汉堡汉密尔顿（HH）阶段〜12-13，并准备进行电穿孔。 </li></ol>准备<ol><li> 质粒米兴<ol><li>每7μLDNA混合物加入1μL0.1％快速绿色（0.1％在18MΩ去离子和蒸馏水中[ddH 2 O]）。 </li><li>对于多重质粒的共电穿孔，以1:1的比例混合质粒。最终DNA浓度应为5-8μg/μL。 </li></ol></li><li> 注射移液器 <ol><li>将移液器拉到微量吸管拔出器上。使用28G注射器针头，用1-2μL质粒填充移液管。 </li><li>使用镊子将移液管吸头切断至10 - 20μm。使用显微镜来帮助确定破碎的移液器吸头的尺寸。将移液器连接到喷雾枪的移液器支架上。 </li></ol></li></ol>开窗注意:窗口过程已在13之前发布。有关其他视觉信息，请参阅参考13。 <ol><li>擦拭所有仪器和工作区域70％乙醇。 </li><li>将所需数量的鸡蛋从孵化器中取出（6-10），离开室温，再次用70％乙醇单独取蛋。除去培养箱后，鸡蛋保持至少2小时。 </li><li>将鸡蛋放在光照射器的顶部，以确定胚胎定位。用铅笔圈住黑暗中心（ 即胚胎）。 </li><li>使用一根19G的针头，在鸡蛋的钝末端戳一个洞。 </li><li>在鸡蛋的尖端旁边绘制圆圈附近的另一个洞。用19G针去除一小块外蛋壳（约16 mm 2 ），然后用镊子取出一小块内蛋壳（约8 mm 2 ）。 </li><li>用配有19G针头的3mL注射器，通过钝头孔从鸡蛋中取出1.5-2.5mL白蛋白。确保将针角向下倾斜45°。 </li><li>用一小块胶带（1厘米×1厘米）覆盖钝头孔。覆盖绘制的圆形d带第二个孔（2.5厘米x 4厘米）。 </li><li>用弯曲的剪刀（切边= 2.5厘米），在圆圈内切一个窗口。剪刀应平行于蛋壳，窗口直径应小于2厘米。 </li></ol> 4.质粒注射<ol><li>打开picospritzer。将压力设置为18 psi，持续时间设置为5μs。打开气囊和灯泡用于解剖显微镜。 </li><li> 将30mL购买印度墨水的1:10稀释的储备溶液混合在无菌磷酸盐缓冲盐水（PBS）中。储存于4°C。注射印度油墨是获得鸡胚更好的可视化的重要一步。 <ol><li>用稀释的印度墨水溶液填充1 mL注射器。安装27G针头并排出注射器中存在的任何气泡。 </li><li>将胚胎插入距离胚胎约2-3毫米的蛋黄膜下，并在胚胎下轻轻注入〜0.2毫升印度墨水。做不会注射超过0.5毫升的印度墨水，因为它可能会降低胚胎存活。 </li></ol></li><li>将窗口蛋放在夹层显微镜下的鸡蛋架上。使用最高放大倍率进行质粒注射部位的最佳可视化。 </li><li>使用镊子，去除注射区域上的膜。听觉脑干感兴趣区域（ 即 NM和NL）来自Rhombomeres 5和6（R5 / R6）。 R5 / R6位于邻近于作为R5 / R6质粒注射的标志的耳朵（发育成内耳的脊椎动物中的胚胎结构）。 </li><li>将DNA填充的移液管放置在覆盖R5 / R6区域的神经管和使用显微操纵器的耳朵之间。理想放置的示意图如图1A所示 。 </li><li>从picospritzer施加空气压力（18 psi，5μs持续时间），以在神经管中排出DNA。 </li></ol>电穿孔<ol><li>打开t他电流/电压刺激器。 </li><li>用PBS注入3 mL注射器，并连接注射器过滤器。在胚胎上加入1-2滴PBS。 </li><li>使用显微操纵器将双极电极降低至胚胎，负极位于注射部位（内侧）上方，正电极位于R5 / R6侧面（ 图1A ）。避免与胚胎直接接触。使用双极电极，其中电极材料是铂铱。使用镊子将尖端之间的间距调整为0.5 mm。 </li><li>使用电流/电压刺激器，以1秒的间隔在50 V脉冲20次脉冲1 ms。 </li><li>电穿孔后，在暴露的区域添加一滴PBS。轻轻取出电极，并用实验室组织和70％乙醇清洗。关闭窗口，用胶带暴露胚胎。 </li><li>标记蛋壳与注入的质粒类型和孵化日期。 </li><li>将鸡蛋放回孵化器，窗口朝上。在38℃下孵育50℃湿度达到所需的发育阶段。 </li><li>如果电穿孔对tet基因载体进行基因表达的时间控制，则每24小时应用多西环素（Dox）以诱导基因表达（参见第6节）。 </li></ol> 6.基因表达时间控制的Tet-on系统<ol><li> 使用以下质粒: pCAGGS-T2TP:表达转座酶的质粒。 pT2K-CAGGS-rtTA-M2:稳定表达Dox结合蛋白的质粒。 pT2K-BI-TRE-EGFP:含有双向tet-on启动子（TRE）的质粒，其表达EGFP报告基因和第二种感兴趣的基因。 注意:上述三种质粒需要以1:1:1的比率共同电穿孔。 </li><li> 储备溶液制备: <ol><li>将Dox溶于无菌PBS中以产生1mg / mL储备溶液。储存于-20°C。 </li></ol></li><li> Dox应用程序: <ol><li>诱导表达n在某些发育阶段的感兴趣的基因，每24小时应用Dox。 </li><li>当应用Dox时，取蛋，打开窗户，将50μLDox吸取到绒毛膜蛋白膜上，关上窗户，将鸡蛋放回培养箱。 </li></ol></li></ol> 7.脑干切片解剖区的准备注意:以下章节（7 - 9）和程序已在14之前发布。有关其他视觉信息，请参阅这些参考。 <ol><li>制备琼脂溶液（40 mg / mL琼脂糖， 即 ddH 2 O中为 4％）。将琼脂溶液倒入培养皿中，使其在室温下固化。将冷藏的琼脂板存放在冰箱中，以便将来在组织切片过程中使用。 </li><li>持续发泡95％O 2和5％CO 2 （pH 7.2-7.4和渗透压:295-310 mOsm / L）的人造脑脊髓液（ACSF）。</li><li>用70％乙醇清洁工作区域和振膜，并用蒸馏水冲洗刀片。 </li><li>使用适当的解剖工具将干净的吸液垫放在工作区域上。 </li><li>将琼脂板从冰箱中取出，切成小琼脂立方体（10mm×10mm×8mm）。使用市售的超级胶将琼脂块胶粘到vibratome切片室的阶段。 </li></ol> 8.分离鸡听觉脑干<ol><li>在录音窗口打开鸡蛋。用手术刀穿刺膜囊并清除鸡胚的头部。 </li><li>用锋利的剪刀剁碎鸡肉。 </li><li>将剃刀刀片尖端稍微靠近眼睛。在头尾到尾部中线切开头骨。根据胚胎的年龄施加轻微的压力。较老的胚胎需要更多的压力。 </li><li>轻轻推开皮肤和羽毛，露出头骨，并验证中线切口。 </li><li>申请压力很大，用剃刀刀片切割头骨的头部。立即将刀片放置在眼睛后面，切割整个头骨和脑组织。 </li><li>使用剪刀在头骨的尾部区域，头部两侧的颈部肌肉稍微前方进行中线到外侧的切口，通过拔除头骨和多余的组织来暴露脑和小脑。 </li><li>使用剪刀将组织切割到脑干上，并清除脑部，自由脱离头骨。 </li></ol> 9.准备脑干切片用于体内电生理或成像<ol><li>通过视觉图形来降低脑干。 </li><li>用剪刀切割花梗去除小脑，暴露第四脑室的地板。 </li><li>使用镊子，从脑干表面去除任何膜组织和血管。 </li><li>要阻止脑干，在四分之一的最底端的水平切割第三脑室，只是尾皮到皮层。使横切通过视网膜垂直，以分离脑干。 </li><li>在琼脂块前面的vibratome舞台上放少量超级胶水。 </li><li>使用镊子提升脊髓的脑干，并将其放置在超级胶水上，朝向侧面向下和背侧朝向颤振片。用实验室纸巾或滤纸去除多余的胶水。 </li><li>将氧合的ACSF倒入振动台。将vibratome刀片设置在20-22°角度。以最大振幅振荡开始振动。将舞台向上移动到刀片，使得组织的顶部与刀片平行。 </li><li>迅速将刀片移动到脑干组织。在刀片与组织接触之前，大大减缓刀片。 </li><li>切片组织具有最慢的前进速度。一旦叶片通过整个冠状组织切片，使用移液管轻轻取出切片/ LI&gt; <li>放下200-300微米，再次切片。重复，直到听觉核的解剖标志变得可见，例如，NL的独特的背侧/腹侧神经纤维区域和NM相对于NL的内侧位置。 </li><li>仔细维护ACFS中的切片。这可以使用温水浴在室温（22°C）或接近生理条件（〜41°C）下进行。注意切片的顺序。第一个切片对应于组织的最尾部区域，最后一个切片对应于最多的分布区域。这粗略地代表了从低频编码区域到高频编码区域的听觉脑干的tonotopic组织。 </li></ol>

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作者没有什么可以披露的。

ovo电穿孔是一种表达或敲低感兴趣的基因以分析体内基因功能的方法8,9 。在鸡胚中，将质粒编码的基因表达到不同的听觉脑干区域中是一种创新的方法。为了确保最佳表达，需要几个关键步骤。首先，只注射其囊肿清晰可见的胚胎。如果卵巢不可见，则胚胎不能在电穿孔的正确发育阶段。第二，较小的移液管尖端使得更容易穿透神经管并导致更少的组织损伤。然?...

声音的快速神经编码对于正常的听觉功能至关重要。这些包括声音定位能力1 ，噪音识别2中的语音，以及其他与行为相关的通信信号3的理解。鸟类和哺乳动物的听觉脑干中的类似神经元是高度专门用于快速神经编码的。其中包括鸡核细胞核（NM），核心层流（NL）及其哺乳动物类似物，前内耳蜗核（AVCN）和内侧上橄榄（MSO） 5 。然而，在听觉脑干中，调节快速神经编码的发育机制知之甚少。因此，研究负责快速神经编码的具体基因是为了更好地了解其在au中的表达，调控和功能是有利的发展。 发展中的鸡胚是研究听觉系统发展基础生物学问题的有效和成熟的研究工具。最近的分子进展已经在发展中的鸡胚中通过表达或敲低感兴趣的基因来解决这些生物学问题，以分析体内基因功能8,9 。研究特定基因的调节作用是理解与听觉缺陷相关的病理学的重大进步。在这里，我们将质子编码基因的ovo电穿孔进入鸡听觉脑干，其中声音的快速神经编码发生10 。通过靶向与菱形体5和6相关的听觉神经祖细胞区域11,12（R5 /R6），我们显示了NM和NL中质粒转染的空间控制。此外，我们通过采用tet-on向量系统显示表达的时间调节。这是在多西环素（Dox） 8存在下表达感兴趣的基因的药物诱导性程序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fertilized white leghorn chicken eggs</td>
			<td>Sunnyside Inc. (Beaver Dam, WI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer</td>
			<td>Parker Hannifin</td>
			<td>052-0500-900</td>
			<td>Picospritzer III, single or dual channel</td>
		</tr>
		<tr>
			<td>Current/voltage stimulator</td>
			<td>Grass Technologies</td>
			<td>SD9</td>
			<td>SD9</td>
		</tr>
		<tr>
			<td>Microfil syringe needles</td>
			<td>World Precision Instruments</td>
			<td>MF28G67-5</td>
			<td>28 Gauge, 67 mm Long, (Pack of 5)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td>Warner Instruments</td>
			<td>64-1280</td>
			<td>MP Series: Non-Electrical Pressure Applications</td>
		</tr>
		<tr>
			<td>Stimulating microelectrode</td>
			<td>FHC</td>
			<td>PBSA1075</td>
			<td>PBSA1075</td>
		</tr>
		<tr>
			<td>Air tank/regulator</td>
			<td>NU Laboratory Services</td>
			<td></td>
			<td>Air dry 300 CF</td>
		</tr>
		<tr>
			<td>Fast green</td>
			<td>Sigma Aldrich</td>
			<td>F7258-25G</td>
			<td>F7258-25G</td>
		</tr>
		<tr>
			<td>Clear plastic tape</td>
			<td>Scotch</td>
			<td>191</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline hyclate</td>
			<td>Sigma Aldrich</td>
			<td>D9891-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg refrigerator</td>
			<td>Vissani Wine Refrigerator</td>
			<td></td>
			<td>13.3-16.1&#176; C (56-61&#176; F)</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Hova-Bator</td>
			<td></td>
			<td>37.8&#176; C (100&#176; F), ~50% humidity</td>
		</tr>
		<tr>
			<td>Dissection scope</td>
			<td>Zeiss</td>
			<td>4.35E+15</td>
			<td>SteREO Discovery, V8 Microscope, 50.4X</td>
		</tr>
		<tr>
			<td>Cold-light source</td>
			<td>Zeiss</td>
			<td>4.36E+15</td>
			<td>CL6000 LED</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Narishige Japan</td>
			<td>Model: MM-3</td>
			<td>2 Micromanipulators</td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td>Sutter Instrument</td>
			<td>BF150-86-10</td>
			<td>Thick-walled borosilicate (dimensions)</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td></td>
			<td></td>
			<td>1 mL, 3 mL</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>BD Precision Glide&#160;</td>
			<td></td>
			<td>27 G x 1 1/4, 19 G x 1 1/2</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Stoelting</td>
			<td></td>
			<td>No. 5 Super Fine Dumont</td>
		</tr>
		<tr>
			<td>Egg holder</td>
			<td>Custom Made</td>
			<td></td>
			<td>Clay base works as well</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td></td>
			<td>Model P-97</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Ultra Cruz</td>
			<td>sc-358811</td>
			<td>PVDF 0.22 &#956;m</td>
		</tr>
	</tbody>
</table>

in ovo electroporation in the chicken auditory brainstem

电穿孔是将感兴趣的基因引入生物相关生物体如鸡胚的方法。长期以来，鸡胚是研究听觉系统发育基本生物学功能的有效研究模式。最近，鸡胚在研究与听力相关的基因表达，调节和功能方面已经变得特别有价值。 在ovo电穿孔可用于瞄准听觉脑干区域负责高度专业化的听觉功能。这些区域包括鸡核细胞（NM）和核层（NL）。 NM和NL神经元来自菱形体5和6的不同前体（R5 / R6）。在这里，我们介绍质粒编码基因的ovo电穿孔研究这些区域的基因相关性质。我们显示一种促进功能表型增益或丧失的基因表达空间和时间控制的方法ES。通过靶向与R5 / R6相关的听觉神经祖细胞区域，我们显示在NM和NL中的质粒转染。通过采用tet-on载体系统可以实现基因表达的时间调控。这是在多西环素（Dox）存在下表达感兴趣的基因的药物诱导性程序。 卵内电穿孔技术 - 与生物化学，药理学和/或体内功能测定一起提供了一种创新的方法来研究听觉神经元发育和相关的病理生理现象。

我们在这里显示， 在ovo电穿孔允许在正常发展的生物系统中的基因表达。质粒编码的基因被重点注射到覆盖R5 / R6的神经管中。 图1A中示出了相对于重要的解剖学标记的电极和移液器放置的示意性示例。在电穿孔后24小时证实质粒注射的正确位置， 如图1B所示 。将质粒靶向注射到R5 / R6上的神经管中，允许在特定的听觉脑干区域（即NM和NL <sup...

Watch this Scientific Journal Video about In Ovo Electroporation in the Chicken Auditory Brainstem at JoVE.com

In Ovo Electroporation in the Chicken Auditory Brainstem

鸟类和哺乳动物的听觉脑干神经元专门用于快速神经编码，这是正常听力功能的基本过程。这些神经元来自胚胎后脑的不同前体。我们提出利用电穿孔技术在鸡胚后脑中表达基因以研究听觉发育过程中的基因功能。

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 ...

in-ovo-electroporation-in-the-chicken-auditory-brainstem

Research

JoVE Journal

Developmental Biology

In Ovo Electroporation in the Chicken Auditory Brainstem

8.4K 次观看.  Northwestern University. 鸟类和哺乳动物的听觉脑干神经元专门用于快速神经编码，这是正常听力功能的基本过程。这些神经元来自胚胎后脑的不同前体。我们提出利用电穿孔技术在鸡胚后脑中表达基因以研究听觉发育过程中的基因功能。 

视频: In Ovo Electroporation in the Chicken Auditory Brainstem

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

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. ...

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 ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...