我们感谢理查德博士河谷和Brikha什雷斯塔，分别在子宫内和体外视网膜电技术，帮助和T.樱井博士对本协议的意见。这项工作是由国立卫生研究院资助F31 NS051008（TJP），POUR LA基金会RECHERCHE Medicale，和人类前沿科学计划（AR）和R01 EY12736（CM）的支持。

第1部分：设置在子宫内和体外视网膜electroporations 1。 在子宫内和体外视网膜electroporations <ul><li>使用一个电极拉马，准备细尖的微量，打破头（或锥尖），使一个角度来看。 </li><li>准备的DNA解决方案所需的浓度，并添加可视化的快速绿色染料（0.05％），少量注射。我建议5μL为母亲的DNA溶液在子宫内的视网膜electroporations每胚胎头部和1μLDNA体外视网膜electroporations解决方案。包括一个GFP的表达结构（或其他荧光蛋白），以可视化转的神经元。 </li><li>通过高压灭菌消毒的手术器械。 </li><li>准备与以下的比率（〜0.2毫升，为每一个怀孕的女性）甲苯噻嗪氯胺酮麻醉剂混合：一个1.0:0.2:4.6的体积比100微克/毫升氯胺酮：100微克/毫升甲苯噻嗪：生理盐水。可用于其他麻醉药。 </li></ul> 2。仅在子宫内对于视网膜electroporations <ul><li>打开电热垫和尿布垫覆盖。 </li><li>在磷酸盐缓冲液（PBS）和加热垫暖中，准备antimycotic抗生素溶液（1:100）。准备为每个​​大坝1〜2毫升。 </li><li>温水浴放置在无菌过滤PBS。 </li></ul> 3。仅适用于体外视网膜electroporations <ul><li>准备视网膜孵化含氧血清培养基（SFM），气泡，用95％的氧气/ 5％的二氧化碳≥2小时25毫升的DMEM/F12。含氧25毫升培养基，添加0.25克牛血清白蛋白（BSA），250μL它的补充和50μL青霉素，链霉素溶液。混合解决方案，解散牛血清白蛋白，然后无菌过滤器的解决方案。这个解决方案应该是每个电实验新鲜。 </li><li>准备视网膜外植体孵化的可持续森林管理+ 0.4％甲基纤维素培养基：高压灭菌器在一个不小的轰动磁铁的一瓶150毫升0.20克甲基纤维素。准备50毫升可持续森林管理的（如上述，无鼓泡）和蒸压甲基瓶。混合这个解决方案，在4 ° C过夜。无菌过滤解决方案翌日，并储存于4 ° C，该解决方案可以保持〜1个月。 </li><li>视网膜外植体的收获（5）日，视网膜外植体的电镀的准备菜：涂层玻璃底培养皿中，用250μL聚- L -鸟氨酸≥2小时，在37 ° C。 （另外，菜肴可涂用聚- L -鸟氨酸的前一天，在4 ° C孵育过夜）洗碗几次，然后用PBS 200μL的DMEM/F12为10微克/毫升层粘连蛋白外衣菜≥在37 ° C 2小时洗碗用PBS在37多次° C和离开，直到在PBS植电镀。 </li><li>对于边界检测，大衣用多聚鸟氨酸菜以上，然后半盘用荧光标记的蛋白外衣可视化边界为2小时（如的Alexa Fluor 555标记的牛血清白蛋白，1:800） 37 ° C。用PBS清洗，然后添加层粘连蛋白如上所述。 </li></ul>第2A： 在宫内视网膜电注射进入体内的E14.5小鼠视网膜电穿孔的DNA <ol><li> E13.5 - E14.5阶段大坝注入0.15毫升的麻醉剂混合腹腔（IP）。它应该需要3-7分钟，直到母亲已经达到了手术的麻醉平面。充分麻醉鼠标不应该回应捏的hindpaw。 </li><li>在这段时间内，切小块的封口膜和吸管〜5 UL​​上的封口膜，DNA溶液。弯曲，在一个90 °度角的柱塞（金属丝）和插入一把拉住micropippette。使用柱塞，吸进微量（使用解剖显微镜可以帮助这个过程）的DNA溶液。 </li><li>一旦母亲已经达到了手术的麻醉平面，使用电动剃须刀刮脸1〜2英寸地区母亲的腹部，然后将其放在她的背面朝下加热垫。准备用酒精垫擦拭剃光其次是碘垫至少两次。每只眼睛，防止眼睛的角膜溃疡，而她的母亲是在全身麻醉下放置一个眼科兽医软膏下降。 <img src="/files/ftp_upload/1333/1333_v2fig1.jpg" alt="图1" /></li><li>镊子，把握皮肤，并用剪刀沿中线（〜1英寸长的）的垂直切口，通过腹部的皮肤和肌肉，暴露腹膜。钳提起腹膜，并通过这种膜类似的垂直切口，显露腹腔（ 图1） 。然后悬垂与纱布的手术视野，接触和被污染的头发，以防止腹腔内容动物。 </li><li>用钳子拉回来的皮肤和腹膜和使用解剖铲通过切口中提取的胚胎（选择最方便的胚胎）。将2胚胎之间的锅铲轻轻拔出腹腔胚胎链。你可以用手指拉出胚胎链。 从这点出发，用无菌PBS水化保持胚胎* </li><li>继续加热垫的母亲，她在解剖显微镜和按摩，使胚胎视网膜朝上下。我建议从最内侧或外侧的胚胎，使它更容易跟踪胚胎电穿孔。 </li><li>在您的主导手的微管和稳定用另一只手的胚胎。随着微，穿透通过子宫壁及羊膜囊的视网膜。根据微量的清晰度，它可能需要相当数量的穿透子宫壁的压力，但一旦通过，微量将进入胚胎和理想的视网膜。它可以是难以控制和评估微量的深度，因为它进入胚胎。最小的微管运动的量，一旦划破膜，以防止扩大的入口孔，造成泄漏羊水和胚胎死亡。避免在子宫壁刺入血管，因为这会导致出血和胚胎死亡。 </li><li>一旦你有信心，微量进入视网膜，你准备驱逐到视网膜的DNA溶液。慢慢地压低柱塞，同时撤回的微管，确保注入DNA是整个微管的路径，作为微量往往渗透到视网膜深。如果你是在视网膜上，驱逐到extraretinal空间之间的外层视网膜层和视网膜色素上皮（RPE）0.5μL（1μL上的微刻度可以通过测量）。您应遵守充入羊水（ 图1）视网膜和泄漏的绿色染料。 </li><li>电桨应保持在PBS填充的培养皿中，在手术过程。胚胎头的两侧轻轻放在与正极（+）注入眼同一侧的桨桨。应用适量的压力，以稳定的头，但不要按太硬，因为这将弹出的羊膜囊和胚胎死亡的结果。当电极到位，提供5 40 V，50 ms的方波频率在60赫兹。这是不寻常的母亲由于目前逃逸的皮肤和肌肉抽搐。 </li><li>重复步骤7-9为每个胚胎被注入和电穿孔。我通常electroporate 4-8胚胎，每个母亲取决于DNA的量，胚胎数，母亲的病情，麻醉下的金额和时间。另外，多个胚胎，可注射，然后电穿孔，而不是分别注射和电穿孔每个胚胎。 </li><li>电穿孔的胚胎后，使用镊子和抹刀更换成胚胎链回腹腔。倒入腹腔〜2毫升的抗生素antimycotic解决方案。 </li><li>使用止血钳和镊子，缝合腹膜，上切口的前部双结开始和进行一个简单的连续缝合切口后部模式。最后的循环使用，以配合关闭和修剪多余的缝线缝合。 </li><li>主食切口封闭，电梯前部钝钳切口的皮肤，小心分开的皮肤腹膜。订书机的牙齿和周围皮肤压低订书机。继续关闭后结束，这通常需要5-7订书钉装订切口。酒精垫擦拭伤口周围的主食。 </li><li>让母亲恢复加热垫（通常需要15-90分钟之间，她唤醒），当她开始翻身起来，她放置在一个新的笼子腹部一侧。 </li><li>为了减少疼痛和不适，母亲收到后觉醒丁丙诺啡（0.05-0.1毫克/公斤，SC），并在稍后的时间点，如果持续不舒服。为了防止脱水，注入1.0 ml生理盐水皮下（SC）〜2-4个小时手术后，并再次，如果晚上和第二天如果需要母亲。手术后24小时，母亲应恢复正常的行为，饮酒和进食定期。 </li></ol>部分2B： 在子宫内电穿孔视网膜视网膜电检索E18.5 <ol><li> 4％多聚甲醛（PFA），准备在PBS〜5毫升每胚胎。可以做一个基于重力输液系统或注射泵灌注。 （另外，E18.5头可以简单地沉浸在4％煤灰一夜之间。） </li><li>注入母亲0.2毫升的麻醉剂混合IP，并确保她是完全根据麻醉。使用剪刀切开皮肤和下面的主食腹膜，暴露腹腔和子宫角。 </li><li>取出电穿孔胚胎和针腹侧。通过削减腹部皮肤，腹膜，膈肌和横向部分肋骨，暴露心脏。皮尔斯左心室灌注设置蝴蝶针，并切断与microscissors右心房。开始灌注，并允许煤灰流〜60秒;血液应该退出右心房，如果灌注成功的胚胎应该变得苍白。杀头的胚胎，并在头15毫升锥形瓶收集在4％的煤灰。电穿孔胚胎重复此过程。保持在4％PFA在4 ° C过夜的头，然后在PBS洗了好几天。安乐死通过颈椎脱位时，所有的胚胎收集的母亲。 </li></ol>卸下视网膜<ol start="=4"><li> PBS洗涤后，在视网膜朝上的夹层菜头管脚，沉浸在PBS。删除揭示了视网膜色素上皮视网膜周围的皮肤。使用26G1 / 2针，穿刺腹视网膜镜头，视网膜色素上皮交界处。一个microscissors的一个边缘插入此孔，并通过一个垂直的切口腹视网膜视盘。这将允许你对东方的视网膜，当你删除它。 </li><li>删除与细点钳的RPE。抢在一个钳腹切口的视网膜色素上皮和使用其他钳远离视网膜剥离的视网膜色素上皮专门的交界处，那里的RPE是牢固地附着在视网膜色素上皮镜头。取出的视网膜色素上皮后，用细镊子掌握的镜头，并拉起了取下镜头。取出视网膜，视神经乳头周围放置钳，夹断视网膜。转让视网膜到一个PBS以及填充，保持跟踪视网膜来到这头。 </li><li>重复步骤4-5对侧视网膜，和所有其他的电穿孔胚胎。非注射视网膜作为一个免疫控制。 </li><li>使用荧光解剖显微镜扫描所有视网膜的GFP +细胞（绿色视网膜）。对于所有GFP +视网膜电成功在这个胚胎，这些视网膜和相应的脑（视觉系统完整），可以作进一步的分析（即切片及免疫组化）（ 图1）处理。 </li></ol>第3a部分： 体外视网膜电到E14.5 在体外培养的小鼠视网膜的DNA注射和电穿孔<ol><li>麻醉用0.2毫升的麻醉剂混合E13.5 - E14.5阶段的母亲，并确保她是完全根据麻醉。用剪刀划破皮肤和腹腔暴露胚胎。抬起钳胚胎链，并通过结缔组织削减从母亲完全消除整个胚胎链。放置在冰与DMEM/F12培养皿胚胎链。安乐死的母亲通过颈椎脱位。 </li><li>穿过一个microscissors整个子宫壁。然后使用镊子，删除从胎盘羊膜囊和关闭捏每个胚胎。杀头每个胚胎和收集的DMEM/F12另一个培养皿首脑在冰上。 </li><li>用口吸管或吸管柱塞，填写所需的DNA溶液量的微量（见第1部分）。 </li><li>在这个协议中，DNA将被注入到周边背视网膜。人们可以通过调整DNA注射部位和电极桨的定位目标等视网膜地区。 </li><li>转移一个头，解剖盘，用PBS和脚头背侧。使用镊子，以消除上述视网膜的皮肤。 </li><li>控股的微量〜10 °水平位置（基本上是平行的背视网膜曲率），推动通过的RPE微管，使针尖之间的RPE细胞和视网膜外层层。进入这个空间驱逐〜0.1-0.4μl的DNA溶液;绿色液体应填满整个空间和通孔在视网膜色素上皮渗出观察。重复注射对侧视网膜。 </li><li>小心，但很快消除引脚（保持头沉浸在PBS）的头部周围放置电极桨，桨头的腹侧方的正极（+）。提供40 V，50 ms，在60赫兹的脉冲，然后将其放在后脑勺与DMEM/F12培养成菜冰（ 图2） 。 <img src="/files/ftp_upload/1333/1333_v2fig2.jpg" alt="图2" /></li><li>所有首长和所有的DNA溶液，重复步骤5-7。在完成electroporations后，添加4井的菜〜1.5 ml的含氧可持续森林管理的解决方案和保持冰（为每个不同的DNA注入解决方案）。 </li><li>传输与解剖盘的DMEM/F12一个头，其中视网膜朝上。腹侧视网膜（或相反一侧的目标区域），用细钳掐撕裂的视网膜色素上皮的孔。通过此孔插入钳子的一端，远离视网膜剥离的视网膜色素上皮，特别是在镜头视网膜交界处。不要切断或损伤视网膜，因为这将导致视网膜到自身孵化过程中崩溃。取出后视网膜色素上皮，使用镊子弹出捏视神经视网膜视网膜基地（离开的镜头完整）。含氧可持续森林管理的转移视网膜。翻转头部和重复对侧眼。 </li><li>电穿孔头完成步骤9。在37℃孵育40-48小时含氧可持续森林管理的视网膜。 </li></ol>第3b部分： 体外视网膜电收获和电镀的GFP +视网膜植（后2天，电） <ol><li>所有视网膜转移到培养皿盖子的DMEM/F12从一个含氧的可持续森林管理以及。选择一个视网膜和使用荧光解剖的范围，以可视化绿色荧光蛋白+（绿色）部分视网膜。 </li><li>使用microscalpel和镊子，解剖和丢弃的非- GFP +视网膜的一部分，因此，仍然只有一个GFP +视网膜块。整个夹层之间不断切换荧光灯和定期光专门选择的GFP +视网膜是有帮助的。传输的GFP +视网膜良好的DMEM/F12。 </li><li>从一个井，重复步骤2中的所有视网膜。对于每一个DNA的条件（每个不同的井），用一个新的培养皿和DMEM/F12培养基。 </li><li>这些绿色荧光蛋白+视网膜现在必须分解为较小的电镀视网膜植。根据解剖范围，切成较小外植体与microscalpel的GFP +视网膜大块。植应为长方形，约200-300微米的长度和宽度。收集在一个良好的DMEM/F12所有植。一个GFP +块视网膜产生〜4-10的GFP +视网膜植取决于组织的大小。重复此步骤，为所有的GFP +收获的视网膜区域。 </li><li>准备所有的GFP +视网膜植后，加250μL可持续森林管理+ 0.4％甲基纤维素层粘连蛋白涂层的培养皿（见第1部分）（保存在4 ° C）。转让4-8 GFP的培养皿中，并使用镊子轻轻地安排成多边形，与位于中间的中心和边缘的菜植植植。 </li><li>确定是研资局层外植体的哪一侧。有时RPE晶体保持连接到外层视网膜层，表明对面是研资局的层。此外，研资局方通常有一些细胞碎片，可以在适当的外植体方向的援助。 </li><li>研资局层，使用镊子，坚决抑制外植体的中心，因此，它将坚持以玻璃盖玻片。在镊子外植体的缩进，可能会看到，这是正常的。 </li><li>培养皿上镀所有植后，转移到37℃培养箱。首次观察到视网膜轴突电镀后4-6小时，植维持数天的健康，但24小时后可出现显着繁茂。植，可用于无数在体外实验，用4％PFA固定为30分钟，免疫染色（ 图2） 。 </li></ol>边境含量<ol start="8"><li>另外，转让4 GFP +植边界检测一个已准备的菜（见第1部分）。排列在盘的中心垂直线的外植体，逼近边境的位置。 </li><li>下一个荧光解剖显微镜，板外植体，使他们〜50-150毫米的荧光边界。菜在37 ° C孵育24小时，让充裕的时间，研资局轴突到达边境基板。 30分钟，可以固定在4％的煤灰植免疫（ 图2） 。 </li></ol>代表性的成果<img src="/files/ftp_upload/1333/1333_v2fig3.jpg" alt="图3" />

<ol>
	<li>Chalupa, L. M., Williams, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Eye Retina and the Visual Systems of the Mouse. , (2008).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc Natl Acad Sci U S A. 101, 16-22 (2004).</li><li>Ishikawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+GDNF+gene+transfer+into+axotomized+retinal+ganglion+cells+using+in+vivo+electroporation+with+a+contact+lens-type+electrode.">Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.</a> Gene Ther. 12, 289-298 (2005).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nat Protoc. 1, 2710-2718 (2006).</li><li>Garcia-Frigola, C., Carreres, M. I., Vegar, C., Herrera, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+into+mouse+retinal+ganglion+cells+by+in+utero+electroporation.">Gene delivery into mouse retinal ganglion cells by in utero electroporation.</a> BMC Dev Biol. 7, 103-10 (2007).</li><li>Petros, T. J., Shrestha, B. R., Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+sufficiency+of+EphB1+in+driving+the+ipsilateral+retinal+projection.">Specificity and sufficiency of EphB1 in driving the ipsilateral retinal projection.</a> J Neurosci. 29, 3463-3474 (2009).</li></ol>

在这段视频中，我们已经证明了电技术，无论是在子宫内和体外成胚胎小鼠视网膜神经节细胞的基因传递，在子宫内的视网膜电提供了一个操纵在视网膜神经节细胞的基因表达的机制和可视化体内的轴突预测。让胚胎生存，产后检查这些GFP +研资局的预测允许将自己的目标，外侧膝状体（LGN）体外视网膜电提供更多的控制能力为目标，在体外实验中使用特定的视网膜区域。 在体内和体外分析从这些技术中派生的结合使彻底表征研资局的发展和在其他正常的背景下的一个子集的视网膜神经节细胞的轴突预测。虽然我们的演示着重于研资局轴突导向视交叉，无论是在子宫内和体外视网膜electroporations可以研究如何在基因表达的影响研资局的分化，树突状形态，电生理特性，突触的形成和适当的扰动定位在终止区等有利外膝体和上丘。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BTX Electro Square Porator ECM 830 (with foot paddle)</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>450052</td>
<td>&nbsp;</td>
<tr>
<td>Round tweezer electrodes</td>
<td>&nbsp;</td>
<td>Nepa Gene</td>
<td>CUY650-5 or CUY650-7</td>
<td>&nbsp;</td>
<tr>
<td>Silk Sutures</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>101-2636</td>
<td>&nbsp;</td>
<tr>
<td>Glass micropipettes with plungers</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>5-000-1001-X10</td>
<td>&nbsp;</td>
<tr>
<td>Antibiototic-antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240096</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12 medium</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11330057</td>
<td>&nbsp;</td>
<tr>
<td>Albumin bovine serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8806-5G</td>
<td>&nbsp;</td>
<tr>
<td>ITS supplement</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-1884</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Methyl cellulose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M0512</td>
<td>&nbsp;</td>
<tr>
<td>Glass Bottom Microwell Dishes</td>
<td>&nbsp;</td>
<td>MatTek Corporation</td>
<td>P35G-1.5-14-C</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-ornithine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4957</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells

视网膜和其唯一的输出神经元，视网膜神经节细胞（研资局），包括一个很好的模型来研究生物学问题，如细胞分化，轴突导向，视网膜组织和突触形成[1]。一个缺点是无法以高效率和可靠的操作，尤其是在视网膜神经节细胞在体内的基因表达，否则访问的小鼠视觉通路，。转基因小鼠可用于操纵基因的表达，但这种方法往往是价格昂贵，费时，并能产生有害的副作用。小妞，在OVO电是用来操纵在视网膜神经节细胞的基因表达研究视网膜和RGC的发展。虽然类似的电技术已经开发新生小鼠的幼崽[2]，大鼠[3]，和胚胎小鼠视网膜体外[4]，这些战略没有让研资局的发展和在体内的轴突预测的特性。为此，我们开发了两个应用程序的电， 在子宫内和其他体外之一，专门针对胚胎小鼠视网膜神经节细胞[5，6]。 在子宫内视网膜电，我们可以misexpress或下调特定基因在视网膜神经节细胞， 通过体内的视觉通路，并按照其轴突的预测，使检查指导决策中间目标，如视交叉，或在目标区域，如，外侧膝状体。扰动否则野生型背景的一个子集的视网膜神经节细胞的基因表达，有利于整个视网膜通路的基因功能的认识。此外，我们已经开发出一种伴侣技术分析体外研资局轴突的生长。我们electroporate体外胚胎头，收集和孵化整个视网膜，然后准备从这些视网膜几天后植。视网膜植，可用于多种体外实验，以研究电穿孔研资局轴突指导线索或其他因素的反应。总之，这种技术增强了我们的能力，misexpress或下调基因在视网膜神经节细胞和应大力援助研资局的发展和轴突预测的研究。

Watch this Scientific Journal Video about In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells at JoVE.com

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

在这里，我们目前的操纵基因表达的小鼠视网膜神经节细胞（RGC的）两个技术<em&gt;在子宫内</em&gt;<em&gt;体外</em&gt;电。这些技术使之一，研究如何在基因表达的改变影响研资局的发展，轴突导向和功能特性。

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 ...

in-utero-ex-vivo-electroporation-for-gene-expression-mouse-retinal

Research

JoVE Journal

Biology

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

18.7K 次观看.  Columbia University College of Physicians and Surgeons. 在这里，我们目前的操纵基因表达的小鼠视网膜神经节细胞（RGC的）两个技术在子宫内体外电。这些技术使之一，研究如何在基因表达的改变影响研资局的发展，轴突导向和功能特性。

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

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

E16大鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

科研

生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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 ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.