这项工作是由理化学研究所脑科学协会（BSI），人类前沿科学计划（HFSP）（批到TS）和理化学研究所少年副研究员计划（JRA的，授予MK）理化学研究所脑科学研究所（BSI），人类前沿科学计划（HFSP）（批到TS）和理化学研究所少年副研究员计划（JRA，授予MK）资助了这项工作。

1。毛细血管和DNA注射液的制备<ol><li>纯化质粒DNA的一个最大的预习或等效的方法，使终浓度为2-3微克/μL。分装100微克的DNA，加上快速的绿色染料（1％的股份）和TE中加入10μl（10MM Tris碱，1mm的EDTA溶液，pH值8.0）和调整卷到100μL注射组合的最终DNA浓度为1μg/ μL。质粒DNA的浓度可以改变取决于质粒的大小，而且其转染效率，需要单独进行测试。 </li><li> 5分钟旋转的DNA溶液在室温下14,000 RPM，并收集上清，以消除任何残留。 </li><li>拉玻璃毛细管tubea使用微量拉马的玻璃毛细管。用镊子夹断尖，使针尖直径为20-30微米。 800微米，1毫米，从尖端测量和检查外部直径小于60微米（ 图1A） 。 </li><li>将毛细管的显微操纵器，并填写与矿物油。为了填补毛细管DNA溶液，浸泡到溶液中的提示和吸DNA溶液。 </li></ol> 2。垫条铂电极在这里，我们用棒铂电极（NEPE基因，CUY611P2 - 1）electroporate到肤浅的大脑区域，如皮层（图1F），。 3。微电极的制备使用微电极进入大脑（即，丘脑和下丘脑）（图2B - E），深部电穿孔。 <ol><li>一端的钨（负极）和铂（正极）线盘绕镀金引脚和固定用封口膜。干棉签插入电线和包装两端封口膜（图1B）。 </li><li>均匀磨砺用砂纸线的一角，直到它到达外部直径20-30微米和60微米，800微米1毫米的提示（图1C和D）。 </li><li>应用波兰绝缘电线的指甲薄外套。指甲油干后，用丙酮浸泡棉签除去头（约200微米的尖端）。重要注意事项:为了防止破坏老鼠的子宫中，部分在800微米，1毫米的尖端直径应不超过70微米（图1E）。 </li></ol> 4。手术的制备<ol><li>麻醉孕鼠（E9.5 - E15.5）。我们证明与医药级戊巴比妥钠麻醉（表二50微克每克体重，注入腹腔，并等待5分钟）。巴比妥钠作为麻醉剂的替代品，包括氯胺酮/甲苯​​噻嗪注射或气体麻醉药（气是可取的时间很短程序）。注入应注意做是为了避免任何机关，否则会造成损伤子宫或杀死动物。当成功的IP，成活率95％以上。此外，手术可在露天环境中进行。缺乏动物捏他们的脚趾，以确认麻醉前开始运作，测试。 </li><li>剃须头发从腹部用刀片和50％乙醇。准备与磨砂交替的优碘和酒精（70％）的皮肤。 </li><li>使用细剪刀腹正中线切口，拉出到37的所有子宫角仔细° C预预热的磷酸盐缓冲液（PBS）湿纱布，这是放置在伤口周围。保留子宫湿润，用PBS所有的时间。 </li><li>保持一个灵活的食指和中指之间的光缆和光纤放置在子宫角。清洁电缆与70％的乙醇，使用前。没有显微镜或放大镜是用于可视化的需要。光纤光光缆和拇指之间的位置子宫，并轻轻挤压，以推高接近胚胎在子宫壁上。 </li></ol> 5。注射DNA和电<ol><li>当胚胎的位置，插入玻璃毛细管小心地将目标脑室注入约1μlDNA溶液（图2A）。 </li><li>对于电electoporation坚持铂电极使用的brainIf肤浅的一部分，子宫以外的地方的电极，并根据制造商的指示（见表一）申请提出方波电流脉冲。如果usingFor更深的大脑部分的电，nmicro电，eedle型电极使用。 Iinsert罚款钨成DNA的负电极注入子宫和两个电极之间发生的目标区域心室和铂电极，然后申请提出方波脉冲电流（见表一）（图2B）。 </li></ol> 6。邮政电<ol><li>将其原价的子宫角回端子位置，并添加500 ° C预热的PBS 37μL。 </li><li>缝合手术缝合线和9毫米autoclipauto剪辑密切外层与内层。 </li><li>在2个小时的加热垫的动物，让从麻醉中复苏。如卡洛芬或丁丙诺啡镇痛药，可用于手术后疼痛管理。 </li></ol> 7。代表性的成果: pCAG - EYFP的皮质电的一些例子使用在不同的时间点坚持铂电极构造图图32所示。大脑电穿孔在E13.5，E14.5和E15.5，并在6日龄（P）的收获。 AThe ntibody RORC染色显示，4层的位置和EYFP的转染细胞位置绿色荧光抗体染色显示，两个图像超施加 （图3A和B ）。标记皮层细胞电穿孔的皮层细胞层显然是在每次实验（图32），这表明随时间变化的电皮质层的特定缆/ LOF可能的不同。 胚胎和细胞转染效率的可行性取决于胚胎的年龄和电的位置。样品表1所列，不过，条件应该是优化每个阶段，大脑的一部分取决于电。 进入深部组织，如丘脑和下丘脑使用棒铂电极的电穿孔经常给低效率（见表1）。通过使用微电极whichelectrodes，这将达到大脑深部，可以解决这个问题。图4显示pCAG EYFP到发展中国家间脑电的例子。质粒DNA溶液注入第三脑室E11.5和微电（ 图 3C4C）。电穿孔面积限制在丘脑，其中大部分的轴突项目皮层（图3B4B） 。 EYFP，fillesfills整个神经元轴突投射到皮层的第4层，也可以显示。 （ 图 3A4A）。 <img src="/files/ftp_upload/3024/3024fig1.jpg" alt="图1"/> 图1展示的毛细血管的正确形状 。卡通示意图。 （A）玻璃毛细管拉使用微量拉马做出一定的形状。小费是由钳捏关闭，使其20-30微米。测量的提示（红色支架）800微米- 1MM，并确保外部直径小于60微米（绿色支架）。 （二）钨或白金线的一端是盘绕镀金引脚和封口膜固定。然后用砂纸，以使他们的尖端成为20-30微米的外部直径60微米和1毫米的尖端。 （三）钨丝之前塑造的形象。 （四）钨丝后塑造的形象。 （五）后申请的指甲油薄薄一层图像的钨丝。在E比例尺是10微米（最小刻度）为行政长官。 （六）坚持铂电极（NEPA基因。CUY611P2 - 1）。在F比例尺为2毫米。 <img src="/files/ftp_upload/3024/3024fig2.jpg" alt="图2"/> 图2。外科手术的卡通模式。 （一）图注入侧脑室或大的胚胎（左侧）， 进入第三脑室或小胚胎（右侧） 。 （二）图electrporation铂棒电极（左侧）和针型电极（右侧）。 <img src="/files/ftp_upload/3024/3024fig3.jpg" alt="图3"/> 图3。电穿孔与发展鼠标端脑pCAG - EYFP E13.5（一），E14.5（B）和E15.5（C）和P6收获。大脑进行切片的日冕和分别与绿色荧光蛋白抗体或RORC抗体染色和图像合并。 （一）在E13.5转染许多第4层的神经元的电pCAG - EYFP的质粒。与第4层标记RORC重叠。 （二）在E14.5转染的细胞，这是更超过4层神经元表面的电pCAG - EYFP的质粒。 （三）E15.5电标签很多，如2 / 3层浅表神经元。比例尺200微米。 <img src="/files/ftp_upload/3024/3024fig4.jpg" alt="图4"/> 图4。EYFP转成丘脑-皮层的轴突（TCA） 。 （一）在皮质层4中的TCA总站发现绿色荧光抗体染色。 RORC抗体染色是用来揭示一个4层的位置。两个图像是取自同一节和合并。 （二）进入丘脑电的位置也显示绿色荧光抗体染色和RORC染色，这是在丘脑11限制。 （三）卡通丘脑电原理图和TCA投影显示。比例尺200微米。

<ol>
	<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240, 237-246 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Kataoka, A., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Imayoshi, I., Shimogori, T., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes+genes+and+neurogenin+regulate+non-neural+versus+neural+fate+specification+in+the+dorsal+telencephalic+midline.">Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline.</a> Development. 135, 2531-2541 (2008).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+application+with+in+utero+electroporation+in+mouse+embryonic+brain.">Gene application with in utero electroporation in mouse embryonic brain.</a> Development Growth &#38; Differentiation. 50, 499-506 (2008).</li><li>Takahashi, Y., Watanabe, T., Nakagawa, S., Kawakami, K., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+Embryology.">Avian Embryology.</a> Methods in Cell Biology. Vol 87, 271-271 (2008).</li><li>Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-activated+site-specific+recombination+in+mice.">Ligand-activated site-specific recombination in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 10887-10890 (1996).</li><li>Indra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+mutagenesis+in+the+basal+layer+of+the+epidermis:+comparison+of+the+recombinase+activity+of+the+tamoxifen-inducible+Cre-ERT+and+Cre-ERT2+recombinases.">Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases.</a> Nucleic Acids Research. 27, 4324-4327 (1999).</li><li>Nakagawa, Y., O'Leary, D. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+patterned+expression+of+orphan+nuclear+receptor+genes+ROR+alpha+and+ROR+beta+in+developing+mouse+forebrain.">Dynamic patterned expression of orphan nuclear receptor genes ROR alpha and ROR beta in developing mouse forebrain.</a> Developmental Neuroscience. 25, 234-244 (2003).</li></ol>

没有利益冲突的声明。

在这个协议中，我们只描述成一个小胚胎大脑使用不同形状的电极禁区pCAG EYFP转技术的好处。对于不同的启动子转染效率的比较如前所述，这表明细胞类型特异性 1 。不过，也有限制，如转染技术，只有短暂的变化取决于质粒，它不会有任何细胞特异性，这是难以控制转染细胞的数量。 ThereforeHowever此协议，与其他技术相结合，将进一步为不同的操作方法的可能性。首先，纳入细胞特异性启动子的质粒DNA，将使细胞类型，如神经元，神经胶质细胞和星形胶质细胞的特定的转染，。二，自染是短暂的，它是不适合那些希望在以后的生活中的基因的功能分析的科学家。因此，用转座质粒DNA整合到基因组的结合，将其转换成稳定转染8。下一步，采用四面体或TET关闭系统将有可能操纵的时间或在8个神经细胞的细胞类型特异性基因表达。另外，可以结合他莫昔芬诱导的Cre - ER（T）的重组酶9和记者小鼠10本议定书。 这个组织的广泛的辅助，将彻底改变在神经科学中所使用的实验设计。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>裸露的钨丝直径125微米</td><td>上午系统</td><td> 797600 </td></tr><tr><td>裸露的铂丝直径125微米</td><td>上午系统</td><td> 767000 </td></tr><tr><td>棒电极</td><td> NEPA基因</td><td> CUY610P4 - 1 </td></tr><tr><td>玻璃毛细管</td><td> Stoelting </td><td> 50611 </td></tr><tr><td>显微</td><td> KD科学</td><td> KDS310 </td></tr><tr><td>剪刀</td><td> ROBOZ </td><td> RS - 5865 </td></tr><tr><td>脉冲发生器</td><td>上午系统</td><td> 2100型</td></tr><tr><td> 9毫米autoclip </td><td> ROBOZ </td><td> RS - 9260 </td></tr><tr><td>镀金引脚</td><td> WPI </td><td> 5482 </td></tr><tr><td> RORC抗体</td><td>英仙座蛋白质组学</td><td> H3925 </td></tr></tbody></table>

mouse in utero electroporation: controlled spatiotemporal gene transfection

为了了解在发育中的大脑，包括信号分子和轴突导向分子，当地的基因转移或淘汰需要的特定区域基因表达的功能。基因靶向敲或淘汰到局部地区是可能的执行，结合特定的综合招聘考试线，这是费力，成本高昂，耗时。因此，一个简单的转染法，在子宫内电穿孔技术，可以用短的时间内执行，就会得心应手的测试可能的候选基因功能的转基因动物的 1,2代前。此外，针对存在大脑里没有具体的综合招聘考试线在子宫内电领域，并会限制胚胎杀伤力3,4。在这里，我们提出了一个在子宫内电到发育中的大脑目标领域相结合的两种不同类型的简单和方便的基因转移电极的方法。首先，使用胚胎光纤光缆光电缆的独特持有方法，将小胚胎（E9.5）进行有针对性的DNA注射入脑室的解决方案和针式电极插入到目标的大脑区域5,6可见。 ，如大脑皮质区的图案出现在早期胚胎阶段，因此，这些早期从E9.5电作出了巨大贡献，了解整个地区的图案事件。其次，孔插入毛细管毛细血管的精确形状，防止子宫损伤。此外，钨和铂丝电极针的精确形状和削尖的使用砂纸，用指甲油 7，这是非常详细的描述在这个协议的方法绝缘。这种独特的技术允许进入大脑禁区的质粒DNA转染，将使小胚胎被电。这将有助于工作在非常早期的萌芽阶段，细胞分化，细胞迁移，轴突导向的许多科学家打开一个新窗口。此外，这种技术将允许科学家质粒DNA转染到深部的丘脑和下丘脑的地方并不多，特定区域的综合招聘考试行增益功能（GOF）或丧失功能（LOF）分析存在的，如大脑发育。

Watch this Scientific Journal Video about Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection at JoVE.com

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

一个发展到小鼠大脑的基因转移方法是通过使用一个独特的手术方法和特殊形状的电极的描述。这种独特的技术使转染的​​质粒DNA的时间和空间，这将有助于研究脑的发育许多神经学家。

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 ...

mouse-utero-electroporation-controlled-spatiotemporal-gene

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

42.8K 次观看.  RIKEN Brain Science Institute. 一个发展到小鼠大脑的基因转移方法是通过使用一个独特的手术方法和特殊形状的电极的描述。这种独特的技术使转染的​​质粒DNA的时间和空间，这将有助于研究脑的发育许多神经学家。

视频: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

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 ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...