研究以预防失明实验孝行长崎的协助下，这项研究符合眼科及视觉研究中使用动物的ARVO声明。 KJW支持由美国国立卫生研究院资助5T32EY013933和5T32DK007647-20。 VBM是由美​​国国立卫生研究院授予K08EY020530支持。

1。视网膜下注射组装设备<ol><li>购买或制作100微米直径的针从一个玻璃毛细管。这可以手动完成使用萨特P-97的移液管的牵拉或其他类似设备。将被加热的毛细管的端部和拉出，直到达到所希望的直径（100微米）。的较小直径的针可用于基因治疗的载体，但是，这是无损伤的细胞或眼睛的推荐直径为细胞注射。玻璃毛细管针钢针相比，具有非常细的，但钝头，用于可视化的喷射流体和访问到视网膜下的空间，以允许不造成视网膜破损。如果需要使用70％的乙醇，并存储在一个无菌的容器，清洁这些拉针。一个15厘米的无菌组织培养皿用胶带按住针到位是一个方式，以保持注射针的覆盖和在无菌环境中。在这一点上，并发症ETE的设置内的手术室，其中该过程将发生。这是最简单，保持手术室，以便在任何时候都保持无菌状态，尤其是当这个过程完成屏障设施内的所有设备。 </li><li>吸干硅氧烷到注射针和弹出硅氧烷留下沿针的内壁涂层。硅氧烷涂层最大化通过针孔的细胞和病毒，否则会粘的内玻璃毛细管壁的通道。 </li><li>在切割25¾轨距与路厄氏锁定的点设置的采血针后直接（从管中取出针）获取一个约8厘米的无菌塑料管的长度。 </li><li>填写1毫升的子-Q 26 5/8-gauge滑小费用无菌生理盐水的注射器从注射器取下针头。 </li><li>将注射针的塑料管的切断端和在另一附加的注射器的路厄氏锁定结束。 </li><li>吸干无菌生理盐水以填补的死空间在油管和毛细管的针，喷射一些盐水通过针，以确保它被正确地连接无任何泄漏。接着，吸一个小的，大约1微升的量的空气，进入注射针的前端。这将分离的盐水从喷射流体，并提供一个表示结束的喷射流体在外科手术过程的视觉线索。的注射装置应保持在清洁的表面，在手术过程中，用70％乙醇擦拭。此外，在外科手术过程中使用的所有设备应之前和之后，用70％乙醇清洗，应该已经被保持无菌并准备使用的注射针的异常。这些针应设置的手术后，而不是重新使用。 </li><li>最后，吸出含有所需的包装的基因治疗的病毒载体的细胞或干细胞的注射液。这可以通过接受注射溶液的无菌移液管尖端，然后移液无菌细胞培养皿的表面上，该溶液。解决方案现在可以很容易地使用的注射器的注射针吸入。所需的量会有所不同，这取决于对鼠标的年龄。我们的目的是产后5天的小鼠，可注射注射液0.5〜0.8μl的。可以使用较少量，0.5微升或更小，可用于年轻小鼠和较大的量，如1微升，对成年小鼠。病毒滴度或需要确定基于对病毒/细胞被注入，和他们的期望的目标组织的细胞数，因为太多的病毒颗粒或细胞在一个小体积，可导致视网膜细胞毒性。在视频中，可视化的目的，我们已用1.5微升，更大的染料量超过必要注入眼睛。 </li></ol> 2。进入视网膜下腔<ol><li>在无菌手术室，稳定或麻醉的小鼠根据当地批准的的动物处理协议，这将不同年龄的鼠标。常见的麻醉成年小鼠包括异氟醚的使用气体或腹膜内注射0.1 ml/10g体重氯胺酮（100毫克/毫升）和甲苯噻嗪（20毫克/毫升）在无菌生理盐水。对于围产期小鼠，常见的麻醉技术包括使用异氟醚的气体或cryoanesthesia（低温）。所有的麻醉技术，必须由您当地的IACUC委员会批准后方可使用。我们通常使用产后一天（P）5只小鼠，虽然类似的技术可以应用到小鼠年轻P0或成年小鼠。外科手术之前，不能开始鼠标停止移动四肢，不再响应从头到脚捏的。围产期小鼠，他们会变得明显，颜色较浅的血流量会有所放缓，知道，他们是在完全麻醉状态下的另一个关键。 </li><li>在围产期小鼠，使用"维纳斯"直剪刀米AKE约1.5毫米的切口，沿封闭盖裂。小心切的凹口处，自然会打开，映入眼帘的未来。这将确保盖子愈合和正常打开，鼠标的发展过程中。可以用于此目的的一种外科刀片，虽然，我们优选Vannas剪刀，因为它们减少了在此过程中的删余的眼睛的风险。在视频中，用剪刀直接穿刺和切沿睑缘。在学习这门技术，钳，应使用，提起眼睑切割前和减少损害眼睛的风险。 </li><li>拉开眼睑到proptose的眼睛，下方的眼睛保持它proptosed注射用的捏住上眼皮稍微弯曲敷料钳。如果在步骤2中的盖的切口过大时，眼睛将下盖滑回，并防止在注射过程中的适当曝光。对成年小鼠，眼部周围施加轻微的压力与博士埃辛的镊子可以保持的注射眼proptosed。 </li><li>做一个小的巩膜切口或后地球的赤道15度的显微叶片表面划伤。此切口应只有足够大，通过该注射针的前端，但不大于。如果愿意，一个小的，如针灸针针可以用来做切口进入视网膜下腔，而不是显微叶片。在深色的动物，将成为暗棕色色素组织（脉络膜视网膜色素上皮细胞）的切口的点处可见。在更轻的着色或白化小鼠中，可以应用于手术标记笔的显微刀片的前端指示的切口部位离开墨斑。如果明确（玻璃）从切口处的流体回流，刀片放在太深，进入玻璃体腔和治疗病毒或细胞可以进入玻璃体内注射过程中的空间。对于visualizati的目的，我们做了切口，在视频这一点。但是它仍然是可能的，在这种情况下，完成一个视网膜下注射，视网膜脱离引起的喷射流体的存在下，可能无法完全愈合在这种鼠标。因此，必须小心削减注射只眼的外表面，而不是通过在视网膜之前。 </li><li>小心地将注射针插入切口部位和推进平行的外眼壁进入视网膜下腔。推进太接近的外壁或眼内太深，将指示错误的位置注入。理解的针是否是在正确的位置，最好的方法是实践的技术。轻轻色素沉着或白化小鼠的练习，可以帮助进行精确的注射到视网膜下腔。这是因为针和注射流体的这些眼睛内是可见的。 </li><li>轻轻的压力注射器的柱塞和杜松子酒注射所需的流体。如果正确地放置在视网膜下腔穿刺针，温和的背压会被感觉到。如果针头内的玻璃体，将有显着更少的背压和在注射过程中的一些潜在的流体回流。如果有明显的反压，保持针取出针管慢慢前至少一个额外的15秒。这将允许高眼压的均衡，而不是注射的病毒或细胞回流回退的切口部位。可能被注入到视网膜下的空间，由于在注射过程中所需的压力增加的部分的空气和盐水。应注意不要过度注入空气或生理盐水，因为它可以冲洗掉眼注入病毒或细胞。从存在太多视网膜下的空间注入流体，也有可能会造成永久性的视网膜脱离。可视化的目的，多余的染料和注入空气，整数Ø在视频中视网膜下腔。的染料会被刷新退了出去，如果过多的视网膜下腔注入，可以清楚地看到，而且气泡保持在视网膜下的空间并没有消失，进入玻璃体。这是用来强调的是，这些注射剂的眼睛都在正确的位置，所以可以想像针应如何进入切口部位。所有注射到视网膜下的空间会导致一个临时的注射流体的存在下，由于视网膜脱离。如果注射过程正确完成，此临时视网膜脱离会在24小时内和小鼠愈合可用于所有的进一步的实验。大约一个星期，由于外科手术的眼睛可能保持柔软，所以，这一段时间期间应避免第二次注射。 </li><li>新生小鼠，用弯曲的敷料镊子轻轻地按压眼球的后面的盖子，并进入预定轨道。它可能是有用等待一分钟，以便让病毒定居在视网膜下的空间，而不是导致其被刷新的切口部位时，轻推眼进入轨道。手术后，一滴0.5％布比卡因（腔投与局部麻醉药 - 麻佳因）从一个25号针头在无菌生​​理盐水稀释至0.25％可以被放置在注射部位。遇险的小鼠没有明显的迹象显示视网膜手术后，已经有足够的前三年使用布比卡因作为一种长效的局部止痛。的小鼠应始终被监控在手术后寻找任何迹象的困扰，如感染，肿胀，或减少在日常活动的鼠标。如果这些情况发生，应通知兽医和作为腹腔内注射，如丁丙诺啡（0.05毫克/千克），可以进一步的止痛药。 </li><li>根据当地批准的动物处理协议的小鼠从麻醉中醒来。鼠标加热P上应保持广告，以维持正常的体温，因为它从麻醉中醒来。手术过程应小于5分钟即可完成，不计时间接受麻醉的小鼠。因此，加热垫是没有必要在实际过程中，而一个学习的技术，因为该过程可能需要较长的持续时间，但可能需要。不应该被放置在鼠标到一个干净的笼子里，直到它已开始上移动自己的。新生儿，柔和的脚趾掐会帮助他们完全从麻醉中恢复过来，他们应该重新被放回到与母亲的粉红色，运动前。随着时间的推移，应监测小鼠前面列出的任何迹象的困扰。 </li></ol>

<ol>
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没有利益冲突的声明。

此视频技术提供视网膜下注射外科手术成功完成，并且确保被放置在所需的位置，有效地治疗眼科疾病的基因治疗载体或干细胞的说明。这种技术允许RPE或光感受器如视网膜细胞的定位，因为它的基因治疗载体或干细胞衍生的组织放置在这些细胞的周边。以前的方法，其中流体被放置在玻璃体腔的玻璃体内注射，而且必须通过视网膜移植，以针对这些特定领域。玻璃体内注射可降低转导的基因治疗载体的目标感光器或RPE，因为不是所有的病毒颗粒会迁移到正确的位置。此外，干细胞是越来越受欢迎，以替代受损的视网膜色素上皮或感光细胞，这些分化的细胞移植内分视网膜空间。 在此过程中的关键步骤是进入视网膜下的空间，因为这样能确保基因治疗载体或干细胞衍生的组织的正确位置，以便有疗效，随着减少视网膜脱离或损坏到眼睛周围的电位组织。必须小心，提前的时间来解决;是能够定位并到视网膜下的空间注入流体。这是可以做到使用着色染料和白化小鼠通过实践所示，在视频中，由于注射针将不可见色素小鼠眼内。经过实践，研究人员将学习感到压力的差异，当流体被正确地放置在视网膜下腔和舒适的外科手术及时完成。 这个手术有一些潜在的修改。这些注射可以执行对不同年龄的小鼠的基因治疗载体或注入干细胞衍生的组织，虽然可能会有所不同，这取决于年龄的鼠标。应谨慎，以确定多少液体会促进治疗的眼科疾病，但不能延长支队和视网膜受损。拉毛细管针可以被修改，以具有不同的直径。较小的病毒注射针是足够的，但直径较大的可能会更好细胞移植。此外，可以进行手术，对成年小鼠。眼组织密度会大得多，所以新鲜的，锐利的手术刀需要渗透到视网膜下的空间。在所有情况下，可以修改此协议的基础上完成一个成功的视网膜下注射的基因治疗载体或干细胞衍生的组织。 所有的视网膜下注射剂将导致临时注射内的流体的存在下，由于视网膜脱离视网膜下的空间，但这种脱离应在24小时内的外科手术愈合。鼠标可以仔细检查，在显微镜下的任何剩余支队。这是可能的，一些老鼠甚至可能无法自然愈合后，占尽天时地利的注射。所有的老鼠都能够接受的检查和实验后，这24小时的时间段，但是眼睛可能是软由于从注射过程中压力的变化，最多一个星期。因此，这种技术的一个限制是在术后第一周的能力进行二次注射。不可以是可能的另一种注射在术后即刻期间期间，由于视网膜脱离，不愈合手术后的发病率增加。建议等待一个持续时间较长的时间，只要在一个月前，重新注入老鼠的眼睛。 鼠标已经成为一个重要的模式生物，研究眼科疾病和潜在的治疗吨帽子会导致人体临床试验。9-12此过程的第二个限制是，并非所有的细胞DNA载体转染视网膜下注射后，他们将在转基因小鼠模型。然而，这个过程可能会更接近人类不是每一个细胞转染的临床过程中。因此，这种技术可以帮助病毒载体的基因治疗或干细胞来源的组织移植疗法治疗年龄相关性黄斑变性，玻璃体视网膜疾病在人类中建立的可行性。未来的应用领域可以包括使用不同的视网膜细胞启动子和分化的干细胞，甚至是多次注射用相结合的治疗方法。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录 </td><td> 评论 </td></tr><tr><td> 0.8-1.10×100毫米的毛细管（玻璃） </td><td> ，金布尔玻璃公司</td><td> 34502 99 </td><td></td></tr><tr><td>烈焰红唇/褐色微管牵引机</td><td>萨特仪器</td><td> P-97 </td><td>成茂microforge可以用来代替。产品编号MF-900 </td></tr><tr><td> Sigmacote </td><td> Sigma Aldrich公司</td><td> SL2-25ML </td><td>有机硅</td></tr><tr><td> Dubecco的磷酸盐缓冲液，氯化钙和氯化镁</td><td> GIBCO-Invitrogen公司</td><td> 14040-133 </td><td></td></tr><tr><td>安全乐25 3/4G×12";采血车集</td><td> BD真空采血管</td><td> 367298 </td><td></td></tr><tr><td> 1毫升子-Q 265/8G防滑提示注射器</td><td>贝克顿 - 迪金森</td><td> 309597 </td><td></td></tr><tr><td> 0.5-10微升Finnpipette II音量可调移液器</td><td> Fisherbrand </td><td> 21-377-815 </td><td></td></tr><tr><td> 1-200微升自然斜角提示</td><td>美国科技公司</td><td> 1111至1700年</td><td></td></tr><tr><td> Discovery.V8体视显微镜</td><td>蔡司</td><td> MC1500 </td><td></td></tr><tr><td> 60毫米×15毫米风格处理聚苯乙烯细胞培养皿</td><td>康宁公司</td><td> 430166 </td><td></td></tr><tr><td> Vannas直剪刀</td><td> STORZ眼药水</td><td> E3383小号</td><td></td></tr><tr><td>弯曲的敷料钳锯齿精致</td><td> STORZ眼药水</td><td> E1408 </td><td></td></tr><tr><td> 15度显微刀</td><td>厚朴眼科公司</td><td> 091204 </td><td></td></tr><tr><td>氯胺酮</td><td> Ketaset III </td><td> NADA＃45-290 </td><td></td></tr><tr><td>二甲苯胺噻嗪</td><td>劳埃德实验室</td><td> NADA＃139-236 </td><td></td></tr><tr><td>布比卡因（腔投与局部麻醉药 - 麻佳因） </td><td>阿斯利康公司</td><td> N / A </td><td></td></tr><tr><td>丁丙诺啡</td><td> Sigma Aldrich公司</td><td> B9275 </td><td></td></tr></tbody></table>

subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye

视力丧失，影响约3.4万人在美国，在未来数年预计将增加。1，基因治疗和干细胞移植已成为主要的治疗手段，治疗视网膜退行性疾病导致的失明。几种形式的自体移植的年龄相关性黄斑变性（AMD），如虹膜色素上皮细胞移植，已经产生了令人鼓舞的结果，为其他形式的基因和干细胞疗法的人体临床试验已经开始。2这些包括RPE65基因置换Leber氏先天性黑蒙和Stargardt病利用人类胚胎干（ES）细胞中的RPE细胞移植治疗的患者。3-4现在有基因治疗的载体，可用于治疗视网膜疾病的患者的干细胞，它是重要的，以验证这些潜在的治疗在动物模型中，前申请ING他们在人类研究中。该鼠标已经成为一个重要的科学模型测试基因治疗的载体和造血干细胞移植在眼的疗效。5-8在这个视频文章中，我们提出了一个技术基因治疗的载体或干细胞分化为视网膜下腔注入老鼠的眼睛，同时最大限度地减少向周围组织的损害。

一个绘图作参考标记的主要结构，示出的鼠标眼玻璃体腔和视网膜下注射的外科手术（箭头， 见图1）的位置显示的箭头。基因治疗的载体，如的LacZ基因的慢病毒载体（ 图2），可以使用这些位置注入。另外，骨髓间充质干细胞，如小鼠胚胎干细胞（ 图3），也可以在这些位点中的老鼠的眼睛被移植。 1微升由巨细胞病毒（CMV）启动子（1×10 8 TU /毫升，Lentigen公司， 图2A）驱动lacZ报告基因的慢病毒载体注射到C57BL/6J小鼠的视网膜下腔。在6周龄的钝性分离摘除眼球，并浸渍在1毫克/毫升X-gal的溶液（5mM的K 3 Fe（CN）的6，2毫摩尔的MgCl 2，0.02％NP 40，和0。1％脱氧​​胆酸钠）中过夜，在37℃下30分钟，切片截面在2％formaldehyde/0.2％戊二醛的眼睛被固定。LacZ报道基因的表达，检测到的X-gal的产品（蓝色）在约10％的光感受器细胞（ 图2B）。 lacZ的表达和完整的周围组织细胞内的存在表明成功的视网膜下注射在老鼠的眼睛。 C57BL/6J小鼠胚胎干（ES）细胞标记与绿色荧光蛋白（EGFP，威康信托基金会桑格研究所）。这些细胞，然后收集，并悬浮在无菌磷酸盐缓冲生理盐水中，移植到产后天（P）5 C57BL/6J小鼠视网膜下的空间，与约1×10 5个细胞每1微升注入。摘除眼球钝性分离，固定在两个星期的年龄在蔗糖梯度冻结在OCT包埋剂（组织-TEK）。眼睛切片，对<html> ualized在荧光显微镜（蔡司）。 GFP-标记的ES细胞的存在下，可以发现视网膜下的空间内注入老鼠的眼睛（ 图3A）。此外，C57BL/6J-Tyr C-2j的 / J（C2J）的小鼠胚胎干（ES）细胞的电穿孔，黄色荧光蛋白（YFP）和视网膜色素上皮（RPE）细胞在体外分化成的。经过一个月的分化，YFP的标记的RPE​​样C2J ES细胞悬浮在无菌磷酸盐缓冲生理盐水中，注入的P5 C57BL/6J小鼠视网膜下空间。在15周的年龄，鼠标采用现场成像自发荧光的YFP在视网膜内的存在（Spectralis激光扫描共聚焦显微镜检眼镜，海德堡工程， 图3B）。 4286fig1highres.jpg"/&gt; 图1。原理老鼠的眼睛的卡通画描绘了老鼠的眼睛玻璃体和视网膜的注射程序标记（箭头）的网站的结构。 <img src="/files/ftp_upload/4286/4286fig2.jpg" alt="图2" fo:content-width="3in" fo:src="/files/ftp_upload/4286/4286fig2highres.jpg"/> 图2。视网膜下注射病毒载体的基因治疗 。 A.慢病毒载体的地图显示lacZ报告基因的巨细胞病毒（CMV）启动子驱动的。 B.在6周龄C57BL/6J小鼠视网膜下注射在产后第五天后，在视网膜上的lacZ报告基因的表达。研资局，视网膜神经节细胞，INL，内核层，光感受器内段（箭头）; ONL，外核层（感光器）（箭头）; OS，光感受器外节。 图3。视网膜下注射的小鼠胚胎干细胞（ES）。A.为期两周的老C57BL/6J小鼠视网膜下注射后的绿色荧光蛋白（GFP）标记的ES细胞在出生后第五天。使用荧光显微镜（Zeiss）进行注入的眼睛视网膜下的空间内是可见的GFP阳性细胞。绿色，GFP标记的ES细胞（箭头）; RGL，视网膜神经节层; INL，内部核层（ONL），外核层（感光器）; / OS，内部和外部的感光段; RPE，视网膜色素上皮细胞。 B的15周龄的C57BL/6J小鼠视网膜所示使用自发荧光活成像（Spectralis扫描激光共焦检眼镜，海德堡工程）的存在下，黄色荧光蛋白（YFP）标记的RPE​​样的C2J小鼠ES细胞在视网膜内。视网膜下注射RPE-YFP-ES细胞在产后第五天。

Watch this Scientific Journal Video about Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye at JoVE.com

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

这种手术的方法说明了基因治疗的载体和干细胞注射到老鼠的眼睛的视网膜下腔。

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

subretinal-injection-gene-therapy-vectors-stem-cells-perinatal-mouse

Nanyang Technological University

Research

JoVE Journal

Medicine

33.0K 次观看.  Columbia University. 这种手术的方法说明了基因治疗的载体和干细胞注射到老鼠的眼睛的视网膜下腔。

视频: Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

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 ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

医学

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

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

用于访问视网膜下腔的另类和验证注射法<i&gt;通过</i&gt;一经巩膜后路

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...