pCAG gapEGFP质粒是博士S.麦康奈尔（斯坦福大学，加利福尼亚州）的礼物。 pCAG - tdTomato质粒博士M.费勒（伯克利分校，加利福尼亚州）的礼物。我们感谢爱德华Ruthazer博士建议使用一个单细胞标记和安妮Schohl验证两个质粒的Cre / loxP位技术支持战略的试点研究和Crair实验室成员（蒙特利尔，QC）的两个质粒战略。 R01 MH62639（MC），美国国立卫生研究院R01 EY015788（MC）和美国国立卫生研究院P30 EY000785（MC）的支持。

1。成套设备，电穿孔<ol><li> 电极 :我们修改杜蒙＃5镊子作为电极使用。 <ol><li>分开，掰开镊子。 </li><li>焊接线，在每个双管齐下。包装线的连接和绝缘胶布离开暴露插脚的一角约25-30毫米的插头。 </li><li>任何合适的塑料垫片之间的两个插脚（如按钮）提供春风行动，把修改的镊子重新走到一起。 </li></ol></li><li> 电气设备:我们使用的电刺激电和示波器和音频监视器提供的电流脉冲，以确认交付的脉冲波形和数量。 <ol><li>一系列的刺激，这也是连接到脚踏板连接线的钳爪。脚踏板作为一个开关。忧郁时完成对电的电路。 </li><li>连接导线从其他叉子的电刺激。 </li><li>连接示波器和音频监控的刺激。 </li></ol></li><li> 微管和喷油器设置 :我们使用急剧拉玻璃移液管和Nanoinject II喷油系统注入极少量的染料或直接到视网膜的DNA溶液。 <ol><li> 3轴显微式喷油系统。 </li><li>拉一个长锥形和小尖的玻璃吸液管。 </li><li>回填土与矿物油和安全喷油拉吸管（详见Nanoinect II说明书）。 </li><li>使用锋利microdissecting的剪刀一个切吸管尖，创造一个小口（2-3微米） 。 </li><li>填写所需金额注射液通过吸管尖吸管。只要拥有尖端的完整性，它可用于多次注射和动物。 注:i）确保有足够的注射液中的提示，回填矿物油是从来没有进入眼内注入装载。二）Nanoinject II系统和具体的电子设备，在这个视频没有实现RGC的成功标签的关键。也可以与其他设备，如picospritzer和与其他常见的刺激电注射使用标签视网膜神经节细胞。 </li></ol></li></ol> 2。研资局贴标质粒解决方案<ol><li> 标签RGC的小群 :我们用一个CAG启动子（鸡β-肌动蛋白与一个巨细胞病毒立即早期增强子）的控制下的一个构造编码EGFP（〜2-3微克/μL）的增强型绿色荧光蛋白）。 EGFP标记的GAP - 43的棕榈酰化序列定位到细胞膜（mut4EGFP）12（生长相关蛋白43） 。这种结构被称为pCAG - gapEGFP。 </li><li> 对于单细胞标记:我们使用了两种结构的组合。第一个向量是一个CAG启动子驱动Cre重组酶（pCAG CRE，Addgene [剑桥，MA]质粒13775）13，第二个向量包含一个floxed停止录像带，有针对性的EGFP（pCAG LNL - gapEGFP）的膜。 pCAG - CRE（〜0.15-ng/μL）使用浓度低于约1,000-10,000倍pCAG LNL - gapEGFP（1-2μgμL），围（凭借强大的EGFP表达的少数细胞相对低的pCAG的Cre浓度）。 </li></ol> 3。视网膜注射和电穿孔协议<ol><li>产后当天0（P0），小五幼仔低温麻醉，而年龄比小五小鼠腹腔注射（0.7毫升/公斤），氯胺酮（4.28毫克/毫升），甲苯噻嗪（0.82毫克/毫升）的鸡尾酒麻醉acepromazine（0.07mg/ml）14。 </li><li>在炎热的珠灭菌消毒所有的手术器械和电极提示。随后，双方在无菌生理盐水之前使用，以避免对眼睛的热损伤凉爽。 </li><li>将鼠标下一个清扫范围和位置的喷油器。 </li><li>对于年轻小鼠比P14（之前的眼开），手术打开眼睑，沿微解剖春天剪刀 b未来眼睑开放的整个长度。 </li><li>部分使眼球突出眼部周围轻轻地运用压力钳彗星的提示。 </li><li>握住到位的眼睛，轻轻地捏眼球周围的皮肤。 </li><li>调整显微带来接近眼球的吸管。 </li><li>按下脚踏板，它是连接Nanoinject II系统，驱逐注射液数滴，从而验证，不堵塞吸管。 </li><li>有了一个稳定的眼球的手，移动，玻璃吸管的尖端刺穿通过视网膜色素上皮细胞，并进入视网膜显微等。 </li><li>注入DESIRED金额按脚踏Nanoinject II系统的解决方案。例如，标签我们做一个单次注射2.3 - 4.6nL单一的视网膜神经节细胞。 </li><li>收回的吸管，小心地将电极提示直接在注射部位，并踩下脚踏刺激完成电路和electroporate眼睛。我们通常使用25V的强度，持续时间50毫秒，1秒，除了设置的方波。 10个脉冲（每个极性脉冲）是适用于年纪比P4和6脉冲（每个极性3个脉冲）为P0到P3小鼠的小鼠。 </li><li>轻轻推眼球放回插座和切眼皮，适用于无菌眼药膏。 </li><li>温度控制的热垫和广场上完全恢复，包括足够的复温及流动的动物，返回他们的母亲的动物。 </li><li>监控动物的任何疼痛，痛苦或不适，如损失的流动性，新郎的姿势异常或故障，的迹象，每12小时。麻醉，注射和电的反复轮不应该执行相同的动物。 </li></ol> 4。注释<ol><li>在本视频中描述的注射方法是一个"瞎子"注射。这是可能的可视化与白化小鼠和彩色解决方案（DII或质粒溶液与快速蓝色微量）工作时注入的数量和位置。然而，注射位置与色素的小鼠株不能直接可视化，因此它是很难口头描述一个人是否已经达到了预期的视网膜位置或应该把吸管具体目标，研资局层多远。因此，我们强烈建议新用户先开始DII白化小鼠注射，因为这些注射可立即在视网膜上的可视化和研资局预测到视网膜和大脑的标签可以用来注射液的质量评估。准备焦距注射10％DII在N，N -二甲基甲酰胺（100％）。一旦此过程中掌握的用户应该能够始终如一的目标有明确的DNA质粒的解决方案，在色素小鼠的视网膜神经节细胞。 </li><li>此影片中所描述可用于注射的方法，使联络DII注射标签的小种群细胞研究retinotopy（4.6nL标签几百 RGC的）14和散装标签RGC的荧光团（Alexa555， 488等）偶联霍乱毒素B亚基研究眼睛特定的隔离，不包括在上面的＃10电一步。一个最大的2 - 3uL总注入量为小鼠比P14的任何解决方案年轻小鼠P14和1 2uL建议。 </li><li>移液器可回填回填之前用矿物油喷油器上安装的吸管，并打破了尖端注入遵循的解决方案。这是特别有用的，当使用高浓度的DNA解决方案（〜6ug/uL），这是一般非常粘稠，难以负荷从吸管尖的。 </li><li>如果玻璃吸管变成堵塞，染料（DII）解决方案的DNA解决方案和乙醇（100％），在水中浸泡棉签仔细擦拭枪头。 </li><li>眼内注射后，收回的眼球枪头，按下脚踏板，再次注入。这是确认的提示没有得到在注射过程中堵塞。如果没有解决方案是配发的，它很可能注射，没有成功。但是，这不应该被作为一个绝对的迹象，注射没有成功。实验者可能再次注入相同的眼，如果需要多个注射部位的细胞数量较大的标签。 </li><li>单RGC的标签，注射速度要缓慢Nanoinject控制箱。这进一步降低注射到视网膜的系统需要更多的时间，在此设置分发的解决方案，同时玻璃吸液管的尖端正在迅速缩回眼球的解决方案。 </li><li>电穿孔产后一天0到3（P0到P3）小鼠需要格外小心，因为它是很容易损伤眼睛（这已成为明显的恢复几天后，当眼睛明显小于正常）。减少脉冲，并施加电压建议为新生儿早期小鼠。眼球后的P4，更具弹性的损害的电。 </li><li>在我们的经验膜有针对性的EGFP或RFP的版本（"gapEGFP"，见12）都远远优于未修改GFP研究retinofugal预测到大脑。然而，pCAG tdTomato，缺乏针对修改的膜，也是行之有效的（图1E）。此外，还可以用于右旋糖酐共​​轭荧光染料使用电协议，上面所述的标签RGC的。 </li><li>通常情况下，会导致9 10注射标记RGCs的双质粒的方法虽然只有约15％的注射结果中只是一个被标记的单研资局。一只眼睛的DNA注射在多个地点或注射到每只眼睛不同的荧光蛋白的质粒编码的数量可以增加成功的个案。 </li></ol> 5。代表性的成果标签是研资局在所有年龄段的观察，从P2到P25的范围，24小时在视网膜神经节细胞后，电EGFP标记，并保持对转染后至少三个星期的表达。 荧光标记的树突（图1A，B）和单一的视网膜神经节细胞的轴突乔木（图1F），可以清楚地可视化和重建。 除了RGC的，这种技术可以用来标记其他细胞类型，如视网膜水平细胞，双极细胞，无长突细胞亚型的各种（图1C） 此方法不干扰正常的视觉地图细化SC（图1E）的正常retinotopy证明的时间当然。 在所有年龄段，在90％左右的情况下，小容量注射剂（〜2.3 - 4.6nL）pCAG - gapEGFP导致在少数视网膜神经节细胞（图1D）的表达。 在大约15％的试验使用的pCAG CRE和每个动物pCAG LNL - gapEGFP结合质粒的单次注射，导致单一的视网膜神经元的标签，包括视网膜神经节细胞（图1A，B，D）和其他类型的细胞，如无长突细胞（图1C）。 <img src="/files/ftp_upload/2678/2678fig1.jpg" alt="图1" /> 图1 - B. EGFP的实例标记，在产后第14天（P13）单视网膜神经节细胞（箭头指向轴突）C.在一个平面贴装视网膜的单爆无长突细胞的例子在P8 D。 EGFP标记包括视网膜神经节细胞和无长突细胞在一个平面安装在P14的。E.背，腹右眼视网膜神经节细胞视网膜视网膜神经细胞群电穿孔和EGFP和时间和腹侧左眼视网膜神经节细胞标记电 ​​镀在P1与tdTomato标记。 F.标记的研资局乔木（2 - D投影）在一个单一的EGFP的例子标记视网膜神经节细胞形成的目标区（星号）可以看出，在SC在P9的地形正确的位置（整个安装，白色的轮廓）。一个矢状切面的SC（250微米厚）（虚线）。清晰，图像中（D）和（六）已转换为灰度和倒置。比例尺（微米）:（一） - （四），（六）:100;（五）:500

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在这个视频中， 我们证明在体内电协议，结果在与编码荧光蛋白质的DNA的结构，产后的小鼠视网膜神经细胞的单个或小群的标签。小群dLGN和SC荧光标记的研资局预测再现类似的投影模式，以前的研究使用亲油性染料研资局的标签，这表明电不干扰正常的研资局轴突乔木细化。我们利用这个协议在单一和不同的小鼠模型的视网膜神经节细胞组与正常，扰乱视觉地图水平的分析视网膜的地图。...

<table border="1"><tbody><tr><th>材料</th><th>公司</th><th>目录编号</th></tr><tr><td>杜蒙＃5镊子</td><td>精细科学的工具</td><td> 11252-20 </td></tr><tr><td>电刺激</td><td>草仪器</td><td> Model S4的</td></tr><tr><td>示波器</td><td>安捷伦</td><td>型号54621A </td></tr><tr><td>音频监听</td><td>草仪器</td><td>型号AM8B </td></tr><tr><td>拉紧器</td><td>萨特仪器</td><td> P - 97型</td></tr><tr><td> Vannas 剪刀 </td><td>世界精密仪器</td><td> 14003 </td></tr><tr><td>微型剪刀b </td><td>特德佩拉</td><td> 1347 </td></tr><tr><td>杜蒙机管局钳彗星 </td><td>精细科学的工具</td><td> 11210-20 </td></tr><tr><td> Nanoinject二系统</td><td>德拉蒙德科学</td><td> 3-000-204 </td></tr><tr><td>玻璃滴管</td><td>德拉蒙德科学</td><td> 3 - 000 - 203 - G / X </td></tr><tr><td>脚踏</td><td>德拉蒙德科学</td><td> 3-000-026 </td></tr><tr><td>矿物油</td><td> Sigma - Aldrich公司</td><td> M3516 </td></tr><tr><td> DII </td><td> Invitrogen公司</td><td> D - 383 </td></tr><tr><td> N，N -二甲基甲酰胺</td><td>西格玛</td><td> D4551 </td></tr></tbody></table>

transfection of mouse retinal ganglion cells by in vivo electroporation

研资局的预测中脑的针对性和细化，是一种流行和​​强大的模型系统，研究开发过程中神经连接的形式如何精确的模式。在小鼠实验中，retinofugal预测被安排在一个地形的方式和形式眼特定层在外侧膝状体，丘脑和上丘（SC）的（dLGN）。 retinofugal预测这些精确的模式的发展通常被标记RGC的人口与荧光染料和示踪剂，如辣根过氧化物酶 1-4研究。但是，这些方法都太粗到个别研资局轴突乔木形态的发展变化，是视网膜地图形成的基础上提供的洞察力。他们也不会允许的视网膜神经节细胞的遗传操作。 近日，电已成为一个有效的方法提供精确的时间和空间控制带电分子传递到视网膜5-11。当前视网膜电协议不允许基因操纵和跟踪retinofugal预测产后小鼠的视网膜神经节细胞的单件或小集群。有人认为，产后体内电穿孔是不是一个可行的方法转染视网膜神经节细胞，因为标签的效率极其低下，因而需要针对胚胎研资局祖细胞发生分化和增殖 6时的年龄。 在这个视频中，我们描述了在体内的基因靶向给药，均有产品，以及出生后小鼠视网膜神经节细胞的荧光右旋糖酐电协议。这种技术提供了一种经济有效，快速参与神经系统发育的几个方面，包括轴突回缩的候选基因的高效筛选和比较容易的平台，分支，层压，再生和突触形成电路开发的各个阶段。总之，我们在这里描述的一种有价值的工具将提供潜在的感官地图发展的分子机制的进一步了解。

Watch this Scientific Journal Video about Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation at JoVE.com

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

我们展示一个<em&gt;在体内</em&gt;电转染视网膜神经节细胞（RGC的）和其他类型的细胞在出生后的小鼠的年龄广泛视网膜的单件或小簇协议。标签和基因操纵产后视网膜神经节细胞的能力<em&gt;在体内</em&gt;是一个发展研究的有力工具。

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 ...

transfection-mouse-retinal-ganglion-cells-vivo

Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

15.4K 次观看.  Yale University. 我们展示一个在体内电转染视网膜神经节细胞（RGC的）和其他类型的细胞在出生后的小鼠的年龄广泛视网膜的单件或小簇协议。标签和基因操纵产后视网膜神经节细胞的能力在体内是一个发展研究的有力工具。

视频: Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔:鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...