We would like to acknowledge David O'Brien for his support with data analysis, and all the members of the Canto-Soler lab for their critical discussions. This work was supported by NIH grants EY004859 and EY022631 (MVCS), Core Grant EY1765, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.

在这项工作中所描述的所有程序都按照约翰·霍普金斯大学推荐的动物护理和使用委员会的指导方针进行。 1.提前的时间：的仪器，试剂和菜准备<ol><li>鸡蛋的制备：在鸡蛋孵化器孵化受精卵白来航鸡卵在37.5℃，60％相对湿度为5天。 注：与摇摆的容量可能是标准化和同步胚胎发育有帮助，但摇摆孵化器是不是绝对必要的此协议的结果。 </li><li>质粒制备： <ol><li> （通过使用RNAi技术1,34的例如）根据需要设计质粒构建用于基因过表达或下调。包括一个报告基因（如EGFP，RFP或LacZ的）在构建体只要有可能。 注：某些常用的启动子，如巨细胞病毒，可在时间后变得沉默。该CAG promot尔（鸡β-肌动蛋白启动子与CMV增强子）是一个非常好的选择，提供感兴趣35-37的基因的有效长期表达。 </li><li>准备以1.5微克/微升无菌PBS的浓度的质粒溶液。可选：提前准备的时间质粒的解决方案，并将其存储冷冻，直到准备使用。 注：较高的质粒浓度不导致效率显著增加，而使用较低浓度的质粒降低转染效率1。 </li></ol></li><li>电极： <ol><li>对于阳极使用粗的金尖电极（见表具体材料/设备）。擦拭用70％乙醇。 注：金的绝缘放倒电极，留下大约0.5mm露出，最好尽量减少电流的泄漏。为绝缘材料的信息可以在表中的特定材料中找到。 </li><li>使阴极出了一个2.5 mm的二次B牛长丝，4.5毫米（微量车夫正常使用）。用钳子的帮助下，撤销方箱和拉直丝为长条形。然后，解剖显微镜和用尺子作为指导下，弯曲长丝成方形的“U”形长3毫米的碱和2毫米高的一侧，与另一侧的灯丝的剩余长度（图1 ，C - D）。使用镊子，蘸96％的乙醇和火焰的电极进行消毒。然后附加的电引线具有小鳄鱼夹到电极的较长臂。 </li></ol></li><li>电穿孔室设置： <ol><li>通过切割盖的无菌1.5 ml离心管中，并贴上它，平面向下，至100毫米培养皿使用磁带（图1E）准备电穿孔室。 注意：其他容器中的类似的尺寸会工作的阱的形式;然而，离心管的盖子是方便和高温高压灭菌。该井必须足够大，以容纳一个小鸡眼睛和阴极电极，但没有那么大，以导致浪费质粒溶液。 </li></ol></li></ol> 2. 前OVO电过程<ol><li>准备仪器： <ol><li>消毒微解剖工具（2对大弯莫洛尼钳打开蛋壳，并舀出胚胎，2对细尖弯曲杜蒙镊子取出视网膜色素上皮，一对波恩齿镊去除晶状体和玻璃体）通过浸渍在96％的乙醇和燃烧的技巧。使用湿巾浸渍在70％乙醇清洁清扫范围。在37℃水浴预热瓶无菌钙无镁汉克的平衡盐溶液（CMF-HBSS）的。填写百毫米无菌培养皿中，以3/4的温暖CMF-HBSS。 </li><li>将电室另一块干净的解剖显微镜下，并填写与120微升质粒的解决方案。连接电极电设备，使得确保定制的电极连接到所述负极和金电极放倒到正极。 </li></ol></li><li>胚胎收集： <ol><li>通过擦拭壳湿巾润湿在70％乙醇清洁鸡蛋。保持整个协议的其余部分消毒程序，以避免携带污染物入文化。用大弯钳莫洛尼小心地轻轻拍打上一个鸡蛋的圆形顶部（在气室），然后捏住并拉出壳片打开蛋壳的孔。 </li><li>与另一双钳取出膜，并舀出来的蛋收集胚胎（注意不要损坏胚胎）。将它与CMF-HBSS菜。 注意：根据需要在2.4.4指定收集尽可能多的胚胎以获得视网膜杯电穿孔所需数量</li><li>关键的一步：根据汉堡和汉密尔顿38阶段的胚胎。 注：要获得最佳效果的胚胎必须是在阶段27（图2A）。 </li><li>安乐死使用莫洛尼镊子断头的胚胎。 </li></ol></li><li>获取视网膜杯： <ol><li>用细杜蒙镊子，小心翼翼地剜除眼睛和转移到一个新的CMF-HBSS菜。 </li><li>从每只眼睛，靠近视神经头的后部开始，并用两个细杜蒙镊子，介绍每个镊子的神经视网膜和视网膜色素上皮和精心之间的一个提示解剖出视网膜色素上皮是谨慎，不损伤神经视网膜（图2B）。 注意：CA 2+和Mg 2+的溶液中的将帮助RPE缺乏从神经视网膜更容易脱离。 </li></ol></li><li>电穿孔： <ol><li>设置电穿孔的参数，以便在15伏，50毫秒的持续时间，和950毫秒间隔5平方脉冲。将“U”形的负电极内部电室（离心盖填充编与120微升含质粒的溶液）。 </li><li>使用弯曲的蒙特镊子传送一个视网膜杯电穿孔室（由舀，不按），并将其放置在“U”形电极的内部，与视网膜朝向电极的底部，并朝上透镜后部（ 图2D）。 </li><li>放置阳极，以便它接触到眼睛的前部，旁边的透镜，以及使用该电穿孔仪的脚踏板递送电脉冲。 </li><li>传输电视网膜杯到一个新的培养皿CMF-HBSS，重复电过程与其余每​​个视网膜杯。 注：最多6视网膜杯可以与电不转染效率显著减少同一质粒的解决方案。 </li><li>握住他们波恩齿镊和至T轻轻拉删除​​每个视网膜电杯的镜头和玻璃体他瞳孔开口在保持眼睛代替与弯曲蒙特镊子的背面。 </li><li>请继续按标准程序将电穿孔的视网膜细胞的分离和培养。 注：有关鸡视网膜细胞的分离和培养一个详细的协议是指贝尔加拉和坎托-索勒，2012年，附加文件4月21日在这个协议中的细胞以低密度的聚鸟氨酸包被6孔板或35。 -mm菜（6×10 5个细胞/ 35毫米直径的孔或皿）。使用的培养基是用青霉素/谷氨酰胺，10％胎牛血清（FCS），和亚油酸 - 牛血清白蛋白，在100微克/毫升介质199。将培养物保持在增湿的37℃培养箱，用5％ 的 CO 2。使用该方法，5 - 6×10 6个细胞，通常可以每每个阶段27电穿孔眼获得。 注：在理想情况下，不超过3小时应该胚胎收集和细胞板之间通过，以保证良好的细胞存活并尽量减少压力。观察和进一步分析一个典型的时间是在培养4天，因为此时细胞已经取得了良好的形态学和分子分化和存活空间还很大。 </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene+promoters+for+liver+expression+by+hydrodynamic+gene+transfer.">Evaluation of gene promoters for liver expression by hydrodynamic gene transfer.</a> J Surg Res. 148 (1), 60-66 (2008).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>You, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+promoter-containing+vector+for+direct+cloning+and+enhanced+expression+of+PCR-amplified+ORFs+in+mammalian+cells.">A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.</a> Mol Biol Rep. 37 (6), 2757-2765 (2009).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev Dyn. 195 (4), 231-272 (1992).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

此协议的成功的最关键的一步是选择胚胎的适当阶段。在以前的出版物，一个范围胚胎阶段的，给出了这些培养，典型地通过的孵化或胚胎天的天（ED）所定义;因此通常认为使用ED 5至6 ED胚胎将得到相同的结果。然而，我们已经发现，在阶段27（ED 5），转染效率将是大约22％的总细胞群中，如上所述;但效率会如果使用级28胚胎（ED 5.5），并以12％的在阶段29（ED 6）降低到16％1。其他关键步?...

从胚胎鸡的视网膜分离的细胞培养物已被广泛用于研究感光细胞生物学的各个方面，包括其存活2-9，分化10-12神经突增生13，等等。该系统的优点，由鲁本·阿德勒和合作者研制在80年代和由他和其他团体14-20，完善居住在小鸡作为动物模型21的固有特性。大尺寸小鸡的眼睛，即使在萌芽阶段，为培养大量的材料。此外，当培养物使用胚胎天（ED）的5进行- 6视网膜，55 - 80％的祖细胞的分化为光感受器14,15,18,22,23，和因为在这种动物的光感受器的大约86％是圆锥 24，这些培养物特别适用于研究着眼于这种细胞类型。 我们最近开发板的PED和表征一个简单的技术，其允许细胞在这些培养高效质粒转，从而通过促 ​​进遗传missexpression研究1扩大了该系统的有效性。该技术的发展，从在将提供转染的可接受水平以使基因的功能在细胞中自主方式的学习方法的科学文献的空隙梗。这部分是因为初级神经元培养物是非常难以染25,26。一些以前提供用于此目的的最常用的技术的包括化学转染方法，例如脂质体转染或磷酸钙介导的转染，这导致在3-5％的数量级的效率，并且可以施加相当大的毒性27-32。即使使用的质粒与一种酶报道系统可以通过放大信号规避差转染效率的问题，也不用鉴别细胞特异性的效应，其结果是基于小细胞群，可能不能代表整个的。在小鸡的另一广泛使用的方法，RCAS病毒感染，只适用于增殖细胞，因此不适合本原发性视网膜培养系统 33。 在当前协议鸡胚眼睛摘除在阶段27（ED 5），视网膜色素上皮细胞（RPE）被除去，视网膜杯被放置在电穿孔室中填充有含质粒的溶液，使用电穿孔的定做电极，然后使用标准技术21视网膜解离和培养。优化该过程之后，我们已经能够始终如一地实现转染效率的培养中的细胞和单独的感光体群体内的25％的总数量的22％的量级上，而不会影响的存活和分化的特性培养1。在这里，我们提供详细的概述的协议，以确保该技术的成功和重现这个过程的所有的重要步骤。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ECM 830 Electro Square Porator</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Genetrode, L-Shaped, 5 mm Gold Tip</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0115 model 512</td>
			<td>Gold tipped electrode used as anode</td>
		</tr>
		<tr>
			<td>Polyimide Tubing</td>
			<td>Vention Medical</td>
			<td>custom made</td>
			<td>Internal Diameter: 0.5mm / wall thickness: 0.15-0.2mm. Used for insulating gold tiped electrode</td>
		</tr>
		<tr>
			<td>2.5mm square box filament, 4.5mm wide</td>
			<td>Sutter Instrument Company</td>
			<td>FB245B</td>
			<td>Used to make cathode electrode</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco - Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Moloney forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>Serrated; Slight Curve; 4.5&#34; Length</td>
		</tr>
		<tr>
			<td>Dumont tweezers 5/45</td>
			<td>Roboz</td>
			<td>RS-5058</td>
			<td>Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length</td>
		</tr>
		<tr>
			<td>Bonn micro forceps, 1x2 teeth</td>
			<td>Roboz</td>
			<td>RS-5172</td>
			<td>Tying Platform; 1X2 Teeth, 0.12mm Teeth; 3.75&#34; Length; .3mm Tip Width</td>
		</tr>
	</tbody>
</table>

efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach

从鸡胚视网膜得到的锥感光，丰富文化已经成为不可缺少的工具，为世界各地的研究人员在研究视网膜神经细胞，特别是光感受器的生物。该系统的应用程序超越基本研究，因为它们可以容易地适用于高通量技术药物开发。然而，在这些培养视网膜感光的遗传操作已被证明是非常具有挑战性的，构成一个重要的限制对系统的有效性。我们最近开发并验证一个前卵内的质粒电穿孔技术，由五倍相比其它目前可用的协议增加1视网膜细胞在这些培养的转染率。在该方法中鸡胚的眼睛摘除在阶段27中，RPE被除去，视网膜杯被放置在含质粒的溶液，并电使用容易地构成定做制作电极。视网膜然后分离和培养用标准程序。这种技术可以通过使用质粒驱动的RNAi技术应用于过表达的研究，以及对基因表达的下调，例如，通常实现的转基因表达在感光体人口的25％。本出版物中的视频格式将使这个技术很容易接触到该领域的研究，从而在原发性视网膜文化基因功能的研究。我们还包括了该过程的圆满成功和可重复性的关键步骤的详细描述。

在这里，我们提出了一个简单的协议，用于质粒转染到去核的小鸡视网膜杯后续分离的细胞培养。转染通过电穿孔使用容易使自定义的电极（ - 2图1）来实现的。在这个协议中所描述的参数进行了优化，以获得转染效率，范围20％之间和27（平均为22％）（图3D）。注意的是，对于以上所述的理由，这些结果后在培养4天后进行定量。在这种情况下，转染效率是指细胞表达的?...

Watch this Scientific Journal Video about Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach at JoVE.com

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

鸡胚视网膜细胞培养物是宝贵的工具，用于感光生物学研究。我们已经开发了基于前OVO之前培养的视网膜质粒电高效的基因转移技术。这种技术显着地增加的转染效率比现有的协议，使遗传操作此系统是可行的。

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 ...

efficient-gene-transfer-chick-retinas-for-primary-cell-culture

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

8.1K 次观看.  The Johns Hopkins University School of Medicine. 鸡胚视网膜细胞培养物是宝贵的工具，用于感光生物学研究。我们已经开发了基于前OVO之前培养的视网膜质粒电高效的基因转移技术。这种技术显着地增加的转染效率比现有的协议，使遗传操作此系统是可行的。 

视频: Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

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 ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

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 ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

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 ...

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 ...