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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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?...

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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 ...

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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。 