Name: whole retinal explants from chicken embryos for electroporation and chemical reagent treatments
Uploaded: 2015-09-30T15:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments at JoVE.com

The work was supported by Barncancerfonden (PR2013-0104), Swedish Research Council (12187-18-3), &#246;gonfonden, Kronprinsessan Margaretas arbetsn&#228;mnd f&#246;r synskadade, Synfr&#228;mjandets forskningsfond and St Eriks &#246;gonsjukhus forskningsstipendier.

该协议是根据"指南为协会的实验动物的护理和使用的研究视觉与眼科"，在这些建议进行的。 1.鸡蛋处理和眼收集<ol><li>存储白来航鸡蛋在12-14℃下不超过1周。如果可能，请使用冷藏酒柜储存受精卵。 注:更高的存储温度导致胚胎发育异常，而较低温度增加了死亡率。延长贮存时间增加了死亡率和畸形发展。 </li><li>孵育在37.5℃下的鸡蛋和60％湿度的蛋孵化器轻轻摇动以每24小时5次的频率。 </li><li>从卵孵化在期望胚胎年龄除去温育卵。 注意:鸡胚的年龄被确定为孵育的时间和被表示为胚胎天（E）或作为阶段（ST），根据该系列由汉堡包和汉密尔顿12中所述发育阶段。 </li><li>将所选鸡蛋在层流罩。擦拭用70％乙醇的蛋壳以避免胚胎的污染。 注:进行与无菌溶液无菌条件下的所有程序<url> http://www.jove.com/science-education/5030/making-solutions-in-the-laboratory&quot;lutions </url>和仪器。 </li><li>点击蛋这一边抵住硬表面敲​​开鸡蛋。放置在100毫米培养皿中的内容。用一个小勺子的胚胎移植到含预热（37℃）PBS洗涤，防止干燥组织35毫米的培养皿。 </li><li>把这个包含胚胎组织上在层流罩一双目视觉解剖显微镜35毫米的培养皿。由俭用细镊子颈部杀头的胚胎和丢弃的尸体。 </li><li>用细镊子做一个切口日粗糙口，腹的眼睛。起始于切口部位，撕开围绕整个眼组织。夹断视神经并从眼窝中删除完整的眼球。从相同胚胎，以用作对照或处理过的眼睛收集的左和右眼。 </li><li>用一个小勺子的目光转移到含预热（37°C）1X PBS一个新的35mm平皿。 </li></ol> 2.准备工作全视网膜外植体<ol><li>用细镊子清除所有的组织在眼部周围，包括巩膜原基。 注意:所述的组织将分离容易留下玻璃体和晶状体视网膜与色素上皮完整（图1A-B）。 </li><li>用细镊子夹住的小切口插入色素上皮在眼睛的背部，而不会损坏神经视网膜（图1C）。 </li><li>用细镊子轻轻撕开色素上皮starti纳克在切口部位，而使透镜和连接到玻璃体（图1D）的整个视网膜。保持眼睛在温暖（37℃）的1×PBS以利于除去的色素上皮。 注意:色素上皮可能难以除去，尤其是沿瞳孔周围睫状体，在眼睛的前部区域，并沿着脉络膜裂。因此，一些的色素上皮细胞可能会留在神经视网膜（图1E-F）。 </li></ol> 3.治疗全视网膜外植体<ol start="1"><li>全视网膜外植体电穿孔 注:整个视网膜植电穿孔可以直接步骤2之后进行。 <ol><li>制备含有1培养基:1的DMEM:F12营养混合物，10％FBS，10U / ml青霉素链霉素，5微克/毫升胰岛素，和2mM L-谷氨酰胺。 </li><li>切断1.5 ml的一次性塑料比色皿（12.5毫米×12.5毫米×45米米）（或任何小容器能够保持300-400微升溶液）约8毫米从底部用细刃锯。 </li><li>选择在1xDPBS的DNA质粒稀释至0.1微克/微升的最终浓度。 </li><li>打开电穿孔和设置参数到15 V为ST20 - ST25（E3-E4）视网膜以及20V ST26 - ST27（E5）视网膜。设置脉冲重复序列5的脉冲50毫秒脉冲长度为1秒的间隔。使用可以由脚踏地板上被控制作为双手将需要保持电极以代替脉冲发生器。 </li><li>铂电极（图2A）连接两个桨形（4毫米扁平，圆形直径），以在脉冲发生器的输出插座。 注:在本协议中所使用的铂电极，由大学研讨会上提出。将电极0.1毫米的铂金板"rondelles"（:950铂/ 5铜铂金级）制成。该连接0.8毫米无线重复使用的塑料管是绝缘的。 </li><li>小心地从2.3步使用聚乙烯巴斯德吸管切断以获得合适的烙铁头，开口尺寸为视网膜外植体顶端的比色皿（或相当小容器）转让全视网膜外植体。避免使用枪头为视网膜容易附着在塑料和撕裂。 </li><li>使用100微升吸管轻轻地去除整个1X PBS周边视网膜外植体，注意不要接触视网膜，避免撕裂。 </li><li>加入100微升的质粒溶液，以反应杯以覆盖整个视网膜外植体。轻轻下压视网膜外植体用钳子，如果它浮在顶部。使用镊子或电极来定位透镜面对反应杯和视神经的任一侧，以比色皿的底部。 </li><li>放置在正极中的透镜和视网膜（图2A）的后面的负电极的前面，而不接触所述组织。 </li><li>通过按下脚踏施加的电流。检查气泡在电极上表示成功出院。 </li><li>使用100μl的移液管除去从试管所有质粒混合，而不会损坏视网膜。所述质粒组合可以重复使用的三到五倍。 </li><li>填满预温（37℃）的1×PBS的试管。使用镊子从试管的壁轻轻分离视网膜外植体。电穿孔后视网膜有时覆盖有气泡，这使得它粘附在反应杯的壁。 </li><li>使用聚乙烯巴斯德吸管向整个视网膜外植体转移到含有1ml预热（37℃），每孔培养基中的24孔平板。每孵育井一视网膜外植体。 </li><li>孵育视网膜外植体在37℃下在旋转器上摇动，用50rpm的恒定速度，细胞培养箱，用5％ 的 CO 2 24小时内。旋转是，以确保最大暴露于介质和preve视网膜至24孔板的底部nt的粘合性。 </li><li>继续执行步骤4。 </li></ol></li><li>全视网膜外植体的化学试剂处理 注:化学处理的整个视网膜植可以直接步骤2之后进行。 <ol><li>制备含有1培养基:1的DMEM:F12营养混合物，10％FBS，10U / ml青霉素链霉素，5微克/毫升胰岛素，和2mM L-谷氨酰胺。 </li><li>用小勺子从步骤2.3传输视网膜植于24孔板含有1ml每孔预温（37℃）的培养基。每孵育井一视网膜外植体。 </li><li>加入化学试剂或媒介物之前孵育整个视网膜外植体60分钟，在37℃在旋转器上摇动，用50rpm的恒定速度，细胞培养箱，用5％的CO 2的内部。 </li><li>制备的化学溶液，例如CDK1 / 2抑制剂III，M相进展13的抑制剂，在最终为30μm的DMSO浓度。 </li><li>除去24孔板，包含整个视网膜外植体，从细胞培养箱。直接添加10μl的化学试剂或载体（DMSO）给视网膜介质，导致为300nM CDK1 / 2抑制剂III在0.01％DMSO的最终浓度。手动旋转24孔板以允许均匀分布在溶液中的化学试剂或车辆。 </li><li>孵育视网膜植于37℃在旋转器上摇动，用50rpm的恒定速度，保温箱，用5％的CO 2所需的时间（长达24小时）内。 </li><li>继续执行步骤4。 </li></ol></li></ol> 4.固定和全视网膜Eexplants的冻结<ol><li>除去含有从细胞培养箱的处理的全视网膜外植的24孔板中。 </li><li>用一个小勺子视网膜外植体转移到24孔板含1毫升/冰冷的1XPBS 4％多聚甲醛。 Incuba德在冰上15分钟。 </li><li>用一个小勺子整个视网膜外植体转移到一个24孔平板含有1ml /孔冰冷1XPBS洗涤视网膜。冰上孵育10分钟。 </li><li>用小勺子给整个视网膜外植体转移到24孔板含1毫升/在1XPBS以及冰冷的30％的蔗糖。在冰上孵育1-3小时取决于视网膜的胚胎天。对于ST20 - ST25（E3）视网膜，孵育1小时后，较旧的视网膜需要在蔗糖更长的温育时间。 </li><li>用小勺子给整个视网膜外植体转移到含有约500微升冻结的低温恒温器安装介质的石蜡膜。通过使用小勺子轻轻移动视网膜外植体在冷冻介质删除尽可能多的蔗糖越好。 </li><li>视网膜外植体转移到含有冻结介质的嵌入模具。放置眼以方便日后切片。将载有关于博士全视网膜外植体嵌入模具Ÿ冰，直到媒体被冻成冰。储存在-80℃。 </li></ol>

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The authors declare that they have no competing financial interests.

在这项工作中对于夹层，电穿孔或化学处理，并从鸡胚整个视网膜外植培养一个详细的协议，提出。该协议是简单，快捷，可为高的成功率和可重复性 结果。 全视网膜外植体电穿孔产生大面积细胞表达目的基因构建的。这是很容易正确地定位在电极和到视网膜的特定部分暴露到规定DNA质粒浓度。这种方法允许一种可靠的方法，调查与相比卵电穿孔一个通?...

视网膜是中枢神经系统的一部分，它是，与它相对简单和充分表征的细胞结构，一个流行的用于研究的中枢神经系统的发展模式。鸡胚胎的眼睛是相比于胚胎的其余部分比较大。因此， 在卵内进行实验操作，如注射或电方便，而且作为一个优秀的工具来获取知识有关的视网膜细胞和体内发育生物学。尽管有这些主要优势，胚胎的存活率可低时的实验是侵入性的，如与电穿孔，重复注射，或结合实验操作。 电穿孔DNA质粒的进入蛋内的鸡胚是一个重要的和成熟的技术1。它允许神经元的标签，在中央NERV跟踪细胞命运以及神经脊髓束的OU系统和它允许异位基因表达来分析蛋白质的功能在体内 。该技术已用于神经管2的研究，后脑3和视网膜4。胚胎视网膜卵内电穿孔具有的相关的体内情况的一些实验困难。眼睛的位置，由于胚胎的颅折叠，相对靠近心脏。这样的接近会增加心脏骤停的下列电穿孔的风险，并与胚胎的年龄的风险增加。此外，访问眼，有必要打开胚胎膜，从而增加出血，畸形和随后的存活率下降的风险。当测试和经常优化新的DNA的质粒没有已知的表型的结果，这些限制可能会降低该方法的有效性和功率甚至对于有经验的实验者。作为呈现在这个协议中，的瓦特的培养孔视网膜外植体，其定义为整个神经视网膜与色素上皮除去，是在卵内的方法补充了一种有效的方法。 化学试剂眼内注射是比较容易的卵内进行。然而，神经视网膜内注射试剂的有效和确切浓度可能难以完全控制。所注入的体积可能由于渗漏和注射的确切位点可以影响眼睛内的试剂的两个分布并通过玻璃体的扩散。变异将对时即一种酶抑制剂的剂量反应曲线被确定的结果的解释主要影响;特别是如果效果小及效果的时间窗口很窄。而且，只有一个单一的眼睛可以从每个胚卵实验执行时使用，由于通过血液流潜在的全身效应到对侧眼。研究开发与处理和控制的胚胎可能会导致额外的实验变异之间的个体差异时，年龄匹配是非常重要的。 由于这些原因， 卵内的方法的当然基于来自鸡胚视网膜植被开发，其中，所述视网膜神经可暴露于均匀的和体外控制的实验条件下。本协议是根据以前的协议5-9开发。来自阶段视网膜外植体（ST）20（胚胎天[E] 3）〜ST31（E7）鸡胚进行解剖，培养和电穿孔以限定的DNA质粒浓度或暴露于包含一个限定的化学试剂的浓度的介质。这里介绍的协议已经成功地实施了最近的出版物，使用几种不同的化学试剂，包括DNA损伤通路的调节，如KU55933，SB 218078和NSC 109555 DITOsylate，和细胞周期，如CDK1 / 2抑制剂III 10,11。

<table><tbody><tr><td>1xPBS (tablet)</td><td>Life technologies</td><td>18912-014</td><td></td></tr><tr><td>10x DPBS</td><td>Life technologies</td><td>14080-048</td><td></td></tr><tr><td>100 mm Petri dish</td><td>VWR</td><td>734-0006</td><td></td></tr><tr><td>100 &amp;mu;l pipette tips</td><td>VWR</td><td>613-0798</td><td></td></tr><tr><td>1.5 ml disposable plastic cuvette</td><td>Thomas Scientific</td><td>8495V01</td><td></td></tr><tr><td>24-well plate</td><td>Sigma Aldrich</td><td>D7039</td><td></td></tr><tr><td>35 mm Petri dish</td><td>VWR</td><td>391-1998</td><td></td></tr><tr><td>70% ethanol</td><td>Solveco</td><td>1054</td><td></td></tr><tr><td>Cdk1/2 inhibitor III</td><td>217714</td><td>Calbiochem</td><td>300 nM in 0.01% DMSO</td></tr><tr><td>Cell culture incubator</td><td>Thermo Forma</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>41966-029</td><td></td></tr><tr><td>Electrodes</td><td></td><td></td><td>Platina, custom made</td></tr><tr><td>Electro square porator ECM 830</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>F12 Nutrient mix</td><td>Life Technologies</td><td>31331-028</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>Forceps</td><td>AgnThos</td><td>0108-5-PS</td><td></td></tr><tr><td>Freezing medium NEG50</td><td>Cellab, Sweden</td><td>6502</td><td></td></tr><tr><td>GFP expressing DNA plasmid (pZGs)</td><td></td><td></td><td></td></tr><tr><td>Humidified incubator</td><td>Grumbach Brutgeraete GmbH, Asslar, Germany</td><td>8204</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>I9278-5ML</td><td></td></tr><tr><td>L-glutamine</td><td>Life Technologies</td><td>25030024</td><td></td></tr><tr><td>Mounting medium ProLong Gold with DAPI</td><td>Life Technologies</td><td>P36935</td><td></td></tr><tr><td>Paraffin film</td><td>VWR</td><td>291-1214P</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>16005-1KG-R</td><td></td></tr><tr><td>Peel-A-Way embedding mold&amp;nbsp;</td><td>Sigma Aldrich</td><td>E6032</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>PhosphoHistone 3 (PH3)&amp;nbsp;</td><td>Millipore</td><td>06-570</td><td>Dilution 1/4,000</td></tr><tr><td>Platinum electrodes (custom made from &quot;rondelles&quot;)</td><td>Sargenta</td><td>390-R (rondeller)</td><td>Dia: 4mm, 0.1 mm thickness</td></tr><tr><td>Platimun electrodes&amp;nbsp;</td><td>Sonidel</td><td>CUY700P4L</td><td>Dia: 4 mm</td></tr><tr><td>Polyethylene pasteur pipette</td><td>VWR</td><td>612-2853</td><td></td></tr><tr><td>Rotator shaker</td><td>VWR</td><td>444-2900</td><td></td></tr><tr><td>Small spoon</td><td>VWR</td><td>231-2151</td><td></td></tr><tr><td>Sucrose</td><td>VWR</td><td>443815S</td><td></td></tr><tr><td>White Leghorn eggs</td><td></td><td></td><td>Local supplier</td></tr><tr><td>Wine cooler</td><td>WineMaster 24, Caso, Berlin Germany</td><td></td><td></td></tr></tbody></table>

whole retinal explants from chicken embryos for electroporation and chemical reagent treatments

视网膜是显影中枢神经系统的良好模型。眼睛的大尺寸和最重要的是可访问的实验操作卵/体内使鸡胚胎视网膜一种多用途和非常有效的实验模型。虽然鸡视网膜容易靶向卵内通过眼内注射或电穿孔，视网膜内的试剂的有效和确切浓度可能难以完全控制。这可能是由于精确注射部位，泄漏从眼睛或物质的不均匀扩散的变体。此外，畸形和死亡率的侵入性操作，如电穿孔后的频率是相当高的。 这个协议描述一个前卵技术培养来自鸡胚整个视网膜植并提供对视网膜试剂的受控曝光的方法。该原COL介绍如何解剖，实验操作，并从鸡胚培养全视网膜植。外植体可以培养约24小时，并可以以不同的操作，例如电穿孔。的主要优点是，该实验是不依赖于胚胎的生存，并且所引入的试剂的浓度可以变化，并为了确定和优化的有效浓度来控制。此外，该技术具有快速，价格便宜，再加上其高实验的成功率，它保证可重复 结果。应当强调的是，它作为一个很好的补充卵内进行的实验。

这个协议描述了从鸡胚整个视网膜植制备（图1A-F）和培养。此协议已被成功地用于整个视网膜植从ST20（E3）的胚胎ST31（E7）。 DNA质粒成整体视网膜外植体电穿孔允许视网膜祖细胞的标记和追踪或过度表达的不同基因产物。对于电的实验中，色素上皮细胞进行了仔细的从ST25（E4½）胚胎的去核眼中除去。整个视网膜外植体，然后放置在含有该DNA的质粒...

Watch this Scientific Journal Video about Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments at JoVE.com

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

本协议描述的方法来剖析，实验操作和鸡胚培养全视网膜植。当高的成功率，有效性和再现性都需要测试质粒电穿孔和/或试剂的物质， 即，酶抑制剂的效应的外植体培养是有用的。

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 ...

whole-retinal-explants-from-chicken-embryos-for-electroporation

Research

JoVE Journal

Developmental Biology

9.4K 次观看.  Uppsala University. 本协议描述的方法来剖析，实验操作和鸡胚培养全视网膜植。当高的成功率，有效性和再现性都需要测试质粒电穿孔和/或试剂的物质， 即，酶抑制剂的效应的外植体培养是有用的。 

视频: Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

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

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

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

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

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 ...

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 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 ...

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 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 ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

从鸡胚胎的直膜到研究棒到锥细胞相互作用的锥形丰富文化

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

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 ...

鸡胚全视网膜外植体电穿孔和化学试剂处理

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在小鸡视网膜有效的基因转移的原代细胞培养研究: