这项工作是由美国国立卫生研究院R01EY020560 - 01和一个WM凯克青年医学研究奖的学者。作者想感谢他的帮助，在视网膜制剂和注射的成像约瑟夫Bedont。

1。质粒准备为电电所需的DNA浓度为5μg/μl。这通常需要使用一个马克西准备（QIAGEN）或等效的方法，由净化和DNA浓度的工作金额被放大所需的质粒。以下步骤描述了DNA的工作金额的准备。 <ol><li>分装100微克的DNA（从最大的预习或同等学历），并稀释到100μL为便于操纵的音量。 </li><li>加入苯酚;苯酚的量应计算以获得约60％的DNA /体积比（微克DNA总量），即:100微克的DNA（100μL）加67μL，苯酚（100微克:167μL）。充分混匀;不吸管向上和向下，因为这可能导致过量的DNA剪切。 </li><li>旋转在14000 RPM，在室温下5分钟的DNA。 </li><li>收集上清，并使用相同的DNA:在步骤1.2的总体积比混合在步骤1.2和自旋为5分钟，在室温下，14000 RPM加氯仿。 </li><li>收集上清，加入10％的3 M醋酸钠上清体积的DNA溶液，轻轻混匀，并添加2.5 × 100％的乙醇上清液体积。混合颠倒。 DNA应该沉淀的溶液在搅拌。 </li><li> 10分钟14000转，旋转的DNA在4 ° C。 </li><li>与350μL70％的乙醇，在5分钟14000转的旋转冲洗4 ° C。 </li><li>空气干燥的颗粒，然后溶于1 × PBS（细胞生物学级）5μg/μL的DNA。不要overdry的样本，因为这会使它很难溶于至PBS。 </li><li>快速绿色染料（10％的股份）的DNA溶液，染料的终浓度为0.1％，作为一个注射示踪。 </li></ol> 2。视网膜下注射的DNA 立体显微镜的协助下执行以下步骤。一旦实行开眼，切口，注射过程所需的时间小于1.5分钟，这是不正确麻醉小狗恢复足够的时间。使用锋利的30号针头，小心地打开眼，沿切割融合的结合上皮（图1C）。不要用力过猛，这可能会导致切割底层的眼睛。避免切割，因为这会导致出血，可以掩盖注射眼睑交界处的范围之外。 <ol><li>麻醉新生小鼠在冰上几分钟（ 图1A），不埋在冰幼崽，因为这可能导致的死亡率。在冰上的时间长度是从小狗小狗，通常5分钟就足够了，但小鼠应仔细监测，作为个人的反应非常迅速，可能需要更长的曝光，以确保适当的麻醉变量。一爪子掐进行止血检查撤退反射。 </li><li>为了方便的DNA注射到视网膜下腔的眼睛必须先打开。拭注射70％的异丙醇准备（泰科医疗），并确定两个眼皮走到一起（ 图 1B）的熔融结合上皮的眼睛。 </li><li>使用锋利的30号针头，小心地打开眼，沿切割融合的结合上皮（ 图 1C ）。不要用力过猛，这可能会导致切割底层的眼睛。避免切割，因为这会导致出血，可以掩盖注射眼睑交界处的范围之外。 </li><li>为了便于渗透眼睛钝端注射针头使用30 - guage针的尖端小切口在巩膜交界处附近的角膜（图 1E） 。不要渗透得太深，因为这可能会导致镜头穿刺。 </li><li>绘制成33计钝截至注射用注射器梯度测量，每次注射（针外径0.52毫米，内径0.13毫米; Exmire microsyringe;伊藤公司）量单独针0.3μlDNA溶液。针插入切口，直到反对巩膜壁的阻力感觉。要小心，以避免针穿透镜头是通过vitreal室（ 图2B）通过。 </li><li>缓慢注入0.3μlDNA溶液，用食指或拇指压下注射器的柱塞视网膜空间。实验者必须小心地控制柱塞抑郁症的速度，因此，在DNA溶液注射到视网膜下腔率不超过DNA溶液在视网膜下腔（图2C）传播的速率。 DNA溶液是粘稠的，所以重要的是针对反对巩膜壁按得太紧，因为这可能导致在不被注入或不扩散均匀的DNA。一个成功的注入将导致更传播的DNA在视网膜下腔的部分解决方案。视网膜的地区，并没有相应的绿色示踪剂应该是明显的，旋转时，注入动物。 </li></ol> 3。电穿孔<ol><li>电穿孔是使用10毫米直径镊子的电极（型号＃522; BTX的仪器）。为了最大限度地从动物电极的导电性和地方注入眼毗邻的正极电极和非注射眼相邻负极电极之间注入小狗的头，浸泡镊子电极在PBS电极（ 图3B）。 </li><li>五，使用脉冲发生器方波:每个脉冲为80伏特，持续50毫秒，950毫秒脉冲之间的时间间隔。 </li><li>电穿孔幼崽现在必须预热，直到他们从冰麻醉恢复。这是可以做到变暖灯或置于幻灯片温暖的顶部的幼崽。如果使用幻灯片温暖，确保适当的垫之间的金属表面，并恢复小狗。痊愈后返回电穿孔幼仔他们的母亲。 </li></ol> 4。分析眼睛的制备<ol><li>电穿孔视网膜的收获和分析的时间进行实验的目标。 GFP的表达可以是严重的可视化后第3天电。 cryoembedding和切片的步骤如下。 </li><li>牺牲所需的时间点的电穿孔动物。 Disect电穿孔眼和固定在4％多聚甲醛于4℃50分钟的彗星。 </li><li>小心地从眼睛的视网膜微解剖远离巩膜，角膜，晶状体，脉络膜和视网膜色素上皮细胞的。可剥离的视网膜下荧光分析粗暴确定的电效率。传输视网膜30％的蔗糖溶液中过夜，4 ° C。 </li><li>冻结视网膜到10月冷冻嵌入媒体（樱花仁昌美国）和存储在-80 ° C。视网膜现在可以切片cryotome到玻片上捕获。 </li></ol> 5。代表性的成果: P0新生儿视网膜与pCAG - EGFP表达质粒电穿孔的例子是在3天以下电（P3）和14天以下电（P13）在图4。电穿孔，视网膜组织的面积是可变的实验，实验在进入视网膜下腔注射和DNA溶液的扩散均匀发生的组织损伤的程度而定。通常90-100％的电穿孔视网膜的功能，已成功注册成立，并表示推出质粒的细胞。然而，在保理业务的组织形态和电效率只有40-60％的电穿孔视网膜时将适合比较分析。花环形成和视网膜脱离针穿透网站几乎总是观察和本地区不应该用于分析。成功的传播过程中的DNA高效电穿孔的针穿透花环和支队的网站组​​织的横向显微注射结果。胶质细胞增生也是一个重要的实验的关注，并几乎总是发生在针穿透网站。然而，与花环形成，视网膜脱离，胶质细胞增生通常是不显着横向电穿孔的组织。如胶质纤维酸性蛋白（GFAP）的免疫组织化学标记，可以用来确定是否在可接受的阈值分析地区的反应性胶质化水平下降。切片视网膜表明电穿孔视网膜形态保持完好和EGFP的表达标签，个别电穿孔细胞。例子是一个P0电（ 图4A - C）和14天以下一个P0电（ 图4D - F），3天以下。 多数在P3的视网膜电穿孔细胞位于视网膜（NBL）在成神经细胞层，并没有表现出任何分化的神经元或神经胶质细胞的视网膜（图4B，C），不同的形态特征。通过P14的电穿孔细胞外核层（ONL）和内核层视网膜（INL）中可以找到。此外，现在的各种标记细胞显示有区别的神经元的特性，包括interneurons的INL，感光器，和Müller胶质细胞（图4E，F）的形态特点。 <img src="/files/ftp_upload/2847/2847fig1.jpg" alt="图1"/> 图1:一个新生（P0），鼠标小狗眼视网膜下注射DNA溶液开放麻醉。一）新生儿小狗放在碎冰麻醉床。对冰眼注入放置。要削减二）融合的结合上皮的位置（B，箭头）眼睑的眼开。 c）切割沿融合交界上皮细胞（ 箭头 ）的眼睑暴露眼球。四）张开眼睛，在融合的交界处切割。 e）一个切口进入眼睛下方角膜巩膜下方，以方便容易插入钝结束的微型注射器。比例尺，B，C，D，E:4MM。 <img src="/files/ftp_upload/2847/2847fig2.jpg" alt="图2"/> 图2。生硬的插入结束进入视网膜下腔微量注射的注射器和注射DNA质粒的解决方案。一）卡通示意图展示微型注射器的正确插入到眼睛和进入视网膜下腔注射器尖端的解决。注射到玻璃体腔的DNA溶液，通过插入插座，或注射进入镜头的注射器通过眼睛将导致失败的实验，如果任何电穿孔细胞数。二）渗透的注射器到在巩膜壁进入视网膜下腔注射器尖端的解决切口。 c）注射进入视网膜下腔的DNA溶液。比例尺，B，C:4MM。缩写如下:的R -视网膜，L型镜头，V型玻璃体，视网膜SRS -空间，截至BES -钝的注射器。 <img src="/files/ftp_upload/2847/2847fig3.jpg" alt="图3"/> 图3。镊子电极到电鼠标的定位。一）卡通示意图展示的镊子电极的电穿孔眼相对的正面和负面的桨方向。绿色代表DNA注入到视网膜下腔的位置。虚线箭头表示带负电荷向正极注入DNA的电泳运动。 DNA电泳出现视网膜下的空间相邻到视网膜，这是面向正极负极。二）积极桨的镊子放在相邻的DNA注射眼和负桨放在相邻的非注射眼。三）对新生小鼠的镊子电极放置的高倍率图像。虚线表示对正极的DNA电泳运动方向。比例尺，C:5MM。 <img src="/files/ftp_upload/2847/2847fig4.jpg" alt="图4"/> 图4。新生小鼠视网膜（P0），电穿孔pCAG - EGFP和分析，在产后第3天（P3）和产后14天（P13） 。 AC）的一个切片视网膜电穿孔在P0 P3 pCAG - EGFP共焦图像。坐在大部分电穿孔细胞在视网膜成神经细胞层（NBL）。 （DF）的一个切片P14的视网膜电穿孔在P0 pCAG - EGFP的共聚焦图像。外核层（ONL）和视网膜内核层（INL） - 电穿孔细胞可以识别。比例尺，自动对焦:50微米。

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在体内电穿孔代表了视网膜细胞DNA的表达质粒转化的快速和有效的方法。此方法允许实验者执行增益功能研究异位引入一个广泛表达的启动子的控制下一个感兴趣的基因或执行使用shRNA结构针对感兴趣的基因功能研究的损失。此外，多个DNA质粒可同时电穿孔，使实验者分析多个基因的影响，或打掉一个基因，同时引入利益的第二个基因。驾驶表达Cre重组质粒电穿孔也可以利用，以消除在时?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>缓冲区饱和酚</td><td> Invitrogen公司</td><td> 15513-039 </td><td> N / A </td></tr><tr><td>氯仿</td><td> JT贝克</td><td> 9180-03 </td><td> N / A </td></tr><tr><td>醋酸钠</td><td> JT贝克</td><td> 3470-05 </td><td> 3个中号的股票</td></tr><tr><td>快绿FCF </td><td>费舍尔生物技术</td><td> BP123 - 10 </td><td> 10％的股票</td></tr><tr><td>异丙醇备考</td><td>泰科医疗</td><td> 6918 </td><td> N / A </td></tr><tr><td> 30 - guage针</td><td>泰尔茂医疗公司</td><td> SG2 - 3013 </td><td> N / A </td></tr><tr><td> Exmire microsyringe </td><td>伊藤公司</td><td> MS * E05 </td><td> N / A </td></tr><tr><td> Tweezertrode（镊子电极） </td><td> BTX的仪器，Genetronics公司</td><td> 522 </td><td> N / A </td></tr><tr><td>电广场Porator（electroporator） </td><td> BTX的仪器，Genetronics公司</td><td>流脑830 </td><td> N / A </td></tr><tr><td>华侨城复合</td><td>美国樱花仁昌</td><td> 4583 </td><td> N / A </td></tr></tbody></table>

in vivo electroporation of developing mouse retina

哺乳动物的视网膜发育过程中基因表达的功能特性仍然是一个重大的挑战。基因打靶生成功能击倒构或有条件的损失仍然是成本和劳动强度大，以及耗时。除了这些挑战，视网膜表达基因可能导致意想不到的困惑以外的视网膜中有重要作用，当使用基因敲除方法。此外，试图找出一个细胞的命运规范和/或终末分化的作用时，能够在增益功能实验的基因异位表达可以是极其宝贵的。 我们提出了DNA质粒的快速和有效地纳入到由新生小鼠视网膜电的方法。短的电脉冲，上述某一个领域在质膜透性的瞬态增加强度 ，促进跨1,2,3,4膜材料转让，应用程序。开创性的工作表明，可以利用哺乳动物细胞的基因转移的方法诱导形成了亲水性的细胞膜，使高电荷的DNA通过脂质双分子层通过5毛孔电。不断的技术发展导致的电作为在体内的基因转移方法，在多个鼠标组织，包括视网膜，这是本文所述6，7，8，9，10的方法的可行性。 DNA溶液注入视网膜下腔，使DNA之间的视网膜色素上皮细胞和视网膜的新生（P0），鼠标和电脉冲，适用于使用镊子电极。鼠标眼睛外侧的位置可以容易镊子电极必要的负面极DNA视网膜正极对齐方向。可确定产后第2天（P2），广泛采纳和转移的基因的表达。由于缺乏显着的视网膜细胞的横向迁移，电穿孔和非电穿孔地区产生。内部组织学控制在适当情况下可作为非电穿孔地区。 视网膜电可以用来表示下一个无处不在的启动子的基因，如冠状动脉造影，或扰乱使用shRNA结构或Cre重组酶基因的功能。更有针对性的表达，可以实现设计与细胞特异性基因的启动子的结构。电穿孔细胞的可视化是通过使用双顺反子结构表达GFP或电穿孔共同构建一个绿色荧光蛋白的表达。此外，多重结构的组合基因的影响或不同基因的功能，同时增益和损失研究可能会被电。视网膜电也可能被用于分析基因顺式调控元件，通过产生适当的表达结构和缺失突变体。可以用这样的实验，找出不足或细胞特异性基因表达11规定的独联体监管区域。潜在的实验是有限的，只有通过构建可用性。

Watch this Scientific Journal Video about In vivo Electroporation of Developing Mouse Retina at JoVE.com

In vivo Electroporation of Developing Mouse Retina

一个执行要么增益或丧失功能的研究目的，方法纳入到小鼠的视网膜细胞的质粒DNA<em&gt;在体内</em&gt;提交。这种方法利用瞬态外部电场的应用诱导细胞质膜的渗透性增加。

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 ...

in-vivo-electroporation-of-developing-mouse-retina

Research

JoVE Journal

Neuroscience

In vivo Electroporation of Developing Mouse Retina

20.7K 次观看.  Johns Hopkins School of Medicine. 一个执行要么增益或丧失功能的研究目的，方法纳入到小鼠的视网膜细胞的质粒DNA在体内提交。这种方法利用瞬态外部电场的应用诱导细胞质膜的渗透性增加。

视频: In vivo Electroporation of Developing Mouse Retina

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

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

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 ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

前全体体内培养，发展的果蝇大脑

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...