在小鸡视网膜有效的基因转移的原代细胞培养研究：<em>前OVO</em>电穿孔法
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 质粒制备： （通过?…
Representative Results
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?…
Discussion
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?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| ECM 830 Electro Square Porator | BTX/ Harvard Apparatus | 45-0052 | |
| Genetrode, L-Shaped, 5 mm Gold Tip | BTX/ Harvard Apparatus | 45-0115 model 512 | Gold tipped electrode used as anode |
| Polyimide Tubing | Vention Medical | custom made | Internal Diameter: 0.5mm / wall thickness: 0.15-0.2mm. Used for insulating gold tiped electrode |
| 2.5mm square box filament, 4.5mm wide | Sutter Instrument Company | FB245B | Used to make cathode electrode |
| HBSS, no calcium, no magnesium, no phenol red | Gibco – Life Technologies | 14175-095 | |
| Moloney forceps | Roboz | RS-8254 | Serrated; Slight Curve; 4.5" Length |
| Dumont tweezers 5/45 | Roboz | RS-5058 | Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length |
| Bonn micro forceps, 1×2 teeth | Roboz | RS-5172 | Tying Platform; 1X2 Teeth, 0.12mm Teeth; 3.75" Length; .3mm Tip Width |
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Citar este artículo
Vergara, M. N., Gutierrez, C., Canto-Soler, M. V. Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach. J. Vis. Exp. (105), e52002, doi:10.3791/52002 (2015).