Name: efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach
Uploaded: 2015-11-02T14:00:00.000+00:00
Duration: 8 min 59 s
Description: 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

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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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+robust+system+for+RNA+interference+in+the+chicken+using+a+modified+microRNA+operon.">A robust system for RNA interference in the chicken using a modified microRNA operon.</a> Dev Biol. 294 (2), 554-563 (2006).</li><li>Nguyen, A. T., Dow, A. C., Kupiec-Weglinski, J., Busuttil, R. W., Lipshutz, G. 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><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: 0.5 mm/wall thickness: 0.15 - 0.2 mm. Used for insulating gold tiped electrode</td></tr><tr><td>2.5 mm square box filament, 4.5 mm 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&quot; 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, 0.12 mm Teeth; 3.75&quot; Length; 0.3 mm 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

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

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 ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

生物学

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

发展中雏鸡光学顶盖中切向细胞迁移的可视化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

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 ...

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.

We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...

在小鸡视网膜有效的基因转移的原代细胞培养研究:

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

鸡胚全视网膜外植体电穿孔和化学试剂处理

在发展中国家小鼠视网膜体内电穿孔

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

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

发展中雏鸡光学顶盖中切向细胞迁移的可视化

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

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

在小鸡视网膜有效的基因转移的原代细胞培养研究:

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

鸡胚全视网膜外植体电穿孔和化学试剂处理

在发展中国家小鼠视网膜体内电穿孔

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

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

发展中雏鸡光学顶盖中切向细胞迁移的可视化

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

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

在蛋和卵外研究中研究鸟类内耳发育的方法