Name: gene transfer into the chicken auditory organ by in ovo micro-electroporation
Uploaded: 2016-04-17T15:00:00.000+00:00
Duration: 6 min 45 s
Description: Watch this Scientific Journal Video about Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation at JoVE.com

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><tbody><tr><td>Sterile 1x PBS pH 7.4</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Fast Green FCF Powder</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>EdU Powder</td><td>Invitrogen</td><td>E10187</td><td></td></tr><tr><td>Click-IT EdU Alexa Fluor 555 Imaging Kit</td><td>Invitrogen</td><td>C10338</td><td></td></tr><tr><td>HiSpeed Plasmid Midi Kit (25)</td><td>Qiagen</td><td>12643</td><td></td></tr><tr><td>ECM 830 ElectroSquarePorator&amp;nbsp;</td><td>BTX Harvard Apparatus</td><td></td><td></td></tr><tr><td>Banana to Micrograbber Cable Kit</td><td>BTX Harvard Apparatus</td><td>45-0216</td><td></td></tr><tr><td>Right Handed &amp;amp; Left Handed Micromanipulators</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>M3301R &amp;amp; M3301L</td><td></td></tr><tr><td>Two 12 mm Magnetic Holding Device Stages with 7 inch vertical posts</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>M10</td><td></td></tr><tr><td>Metal Steel Base Plate 12x24 inch</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>5479</td><td></td></tr><tr><td>Scissors for Eggs 12 cm long curved, 12 mm extrafine blades; Spring Scissors</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>14120</td><td></td></tr><tr><td>Micropippette Puller for pulling needles</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>2.5x2.5 mm Box Platinum Heating Filament</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Glass Capillary Tubes/Needles/No Fiber/Borosil 1 mm</td><td>FHC</td><td>27-30-0</td><td></td></tr><tr><td>Hamilton Glass Syringe 100 &amp;mu;l</td><td>Hamilton</td><td>80601 Model 710LT</td><td></td></tr><tr><td>Mineral Oil (Heavy) for Hamilton Glass Syringe</td><td>Fisher Scientific</td><td>O122-1</td><td></td></tr><tr><td>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><td></td></tr><tr><td>3 cc Disposable Syringes</td><td>Becton Dickinson and Company (BD)</td><td>309657</td><td></td></tr><tr><td>Disposable 21 Gauge Needles</td><td>Becton Dickinson and Company (BD)</td><td>305122</td><td></td></tr><tr><td>One pair of 2 mm Platinum Electrodes</td><td>Bulldog Bio. / Nepagene</td><td>CUY611P3-2</td><td></td></tr><tr><td>Electrode Holder</td><td>Bulldog Bio. / Nepagene</td><td>CUY580</td><td></td></tr><tr><td>One pair of Dumont fine forceps number 5</td><td>Fine Science Tools (FST)</td><td></td><td></td></tr><tr><td>Matte finish invisible tape for sealing eggs</td><td>Office Depot</td><td>520-928</td><td></td></tr><tr><td>Cotton-Tipped Swabs</td><td>Fisher Scientific</td><td>23-400-101</td><td></td></tr><tr><td>Sterile filter tips</td><td></td><td></td><td></td></tr></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

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 ...

科研

发育生物学

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

在OVO的HH阶段10个鸡胚Electroporations

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

生物学

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 ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

神经科学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

在OVO在鸡胚视网膜电穿孔

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

基因转移到老年人的鸡胚<em&gt;前OVO</em&gt;电穿孔

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

基因转移到鸡听觉器官通过

摘要

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此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

在OVO的HH阶段10个鸡胚Electroporations

在奥运电击鸡听觉脑干中

转基因小鼠内耳的发展

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

在微RNA在腹鸡中脑大毛表达

在蛋和卵外研究中研究鸟类内耳发育的方法

在OVO在鸡胚视网膜电穿孔

基因转移到老年人的鸡胚

通过产卵电穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

基因转移到鸡听觉器官通过

摘要

探索更多视频

此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

在OVO的HH阶段10个鸡胚Electroporations

在奥运电击鸡听觉脑干中

转基因小鼠内耳的发展

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

在微RNA在腹鸡中脑大毛表达

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

在OVO在鸡胚视网膜电穿孔

基因转移到老年人的鸡胚

通过产 卵电 穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

在蛋和卵外研究中研究鸟类内耳发育的方法

通过产卵电穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能