Name: cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse
Uploaded: 2016-01-18T14:00:00.000+00:00
Duration: 6 min
Description: Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

伦理声明:小鼠和实验程序的处理被根据用于动物护理欧洲，国内和机构准则进行。 1.在子宫内的电 注: 在子宫内的电进行以前发表12,14。因此，该方法仅在下面的（图1）简单说明。 <ol><li>准备工作<ol><li>制备坚牢绿色无内毒素的先进染同类的质粒（含有1,5- pCAGGS中）溶液如前所述12。 </li><li>拉和研磨（35°角），硼硅玻璃毛细管（直径0.8-0.9）注射。 </li><li>消毒仪器。 </li></ol></li><li>手术<ol><li>在开始手术前施用镇痛剂（卡洛芬，4毫克/公斤体重，SC，24小时，仓库）至少30分钟。 </li><li>保持的长度麻醉到最多35-45分钟（第三阶段-外科麻醉的阶段根据Guedel 15 -最大25-30分钟）。 </li><li>麻醉超时孕鼠异氟醚（诱导室:2.8％，经手术口罩:2.5％）。确认麻醉由无反应到脚趾捏。 </li><li>应用眼膏，以防止在麻醉过程中眼睛干燥。 </li><li>消毒用7.5％Providone碘溶液手术区并覆盖鼠标用无菌纱布（湿润用0.9％苄醇的盐水溶液）与仅暴露手术区。 </li><li>切割用解剖刀打开腹腔（皮肤切口:1.5-2厘米，肌肉切口:1-1.5厘米）。 </li><li>保留从干燥通过用温热0.9％苄醇的盐溶液或其它无菌等渗溶液润湿的指示兽医人员或委员会打开腹腔。 </li><li>轻轻取出子宫角（使用环钳，不要触摸手指子宫S）。 </li></ol></li><li>注射DNA和电穿孔。注:具体细节定位特定的区域显示在第2节。 <ol><li>经由制备的硼硅酸盐毛细管中图1和部分2指示的有色DNA溶液进入心室缓缓注入。 </li><li>通过专门的钳型铂电极（间隔周期长度为50毫秒，间歇暂停950毫秒）应用适当的电压对应的萌芽阶段（例如，36-38 V代表E14）。电压的施加期间脉冲电穿孔桨叶，以尽量减少子宫壁的损伤。 </li></ol></li><li>帖子电/术后护理<ol><li>在腹腔内仔细替换子宫角。 </li><li>缝合肌肉和皮肤切口分开。 </li><li>让我们恢复一热支持设备上的鼠标休息。 </li><li>监督恢复过程中鼠标（行为，饲料水消耗量），4小时，24小时和手术后48小时。如果需要的话，应用额外的镇痛药（卡洛芬，4毫克/公斤体重，24小时，仓库），用于进一步2天。 </li></ol></li></ol> 2.转染特异性脑区注意:正极的位置被示出为通过填充有DNA溶液（右或第三脑室）和正极的位置的心室的垂直中心线之间的角度。当填充左心室中的位置是镜像反转。 图 4中的耳原始的位置被示出，这提供了重要的参考点。 <ol><li>的皮层区转染（图2A，表1）。 <ol><li>如在1.2节进行手术。对于在子宫内电穿孔皮质区域使用E14的胚胎。 </li><li>注入1.5-2微升含4微克的DNA在右侧脑室DNA的溶液通过准备硼硅毛细血管。 <ol><li>小心地用卵圆钳，以便有没有明显的血管穿刺部位上方的位置胚。 </li><li>慢慢注入DNA的溶液从人字缝线约0.75毫米冠状和0.5毫米的横向从矢状缝（图3）。确保插入深度为1.2毫米（E14，从子宫壁测得）。检查用锋利的划分蓝绿色彩。 </li></ol></li><li>通过应用专门钳型铂电极上的电压（间隔周期长度为50毫秒，间歇暂停950毫秒）。 <ol><li>使用3mm或5mm电极桨。 </li><li>使用电压范围从36-38 V. </li><li>放置电极只是前耳原基的中心。 </li><li>从330-0°染retrosplenial皮质使用的角度，转染运动皮层0-40°，转染躯体感觉皮层40-95°，转染梨状皮质95-110°（ 图2A）。 </li></ol></li><li>如第1.4节所述执行后电/术后护理。牺牲小鼠并按照第3条所述电（E18）后分析胚胎4天。 </li></ol></li><li>转染海马（图2B，表1）的。 <ol><li>如在1.2节进行手术。对于在子宫内的电海马结构的使用E15胚胎。 </li><li>注入2微升含4微克的DNA在右侧脑室经由制备的硼硅酸盐毛细管DNA的溶液。 </li><li>通过应用专门钳型铂电极上的电压（间隔周期长度为50毫秒，间歇暂停950毫秒）。 <ol><li>使用3mm或5mm电极桨。 </li><li>使用电压范围为38-40。 </li><li>放置负极只是前耳原基的中心和中心O˚F在耳边原基的中间正极。 </li><li>转染齿状回使用的角度从190-210°，转染大角ammonis（CA）3 210-230°，转染CA2 230-250°，转染CA1 250-275°（ 图2B）。 </li></ol></li><li>如第1.4节所述执行后电/术后护理。如在第3节中所述执行后，完整的海马结构（出生后，P21）分析。 </li></ol></li><li>横向隔核和纹状体的转染（图2C，表1） 注:角度与转染海马重叠。为了有效地转染的横向隔核和没有令人不快的海马染早期胚胎阶段（E12）纹状体必须被使用。 <ol><li>如在1.2节进行手术。对于在子宫内的电外侧隔核和striat的UM使用E12的胚胎。 </li><li>注入1微升含有4微克的DNA在右侧脑室经由制备的硼硅酸盐毛细管DNA的溶液。 </li><li>通过应用专门钳型铂电极上的电压（间隔周期长度为50毫秒，间歇暂停950毫秒）。 <ol><li>使用一个0.5毫米的电极。 </li><li>使用33 V. </li><li>放置电极的中心在耳边原基的水平。 </li><li>从100-170°染横向隔核使用的角度，以转染纹状体170-240°（ 图2C）。 </li></ol></li><li>如第1.4节所述执行后电/术后护理。牺牲小鼠并按照第3条所述电（E18）后分析胚胎6天。 </li></ol></li><li>丘脑和下丘脑的转染（图2D，表1） <ol><li>如在1.2节进行手术。对于在ü泰罗电丘脑和下丘脑使用E12-E13的胚胎。 </li><li>注入含4微克的DNA在侧脑室（左或右）通过制备硼硅毛细管1-1.5微升DNA的溶液。等待2-3分钟，直到溶液扩散进入第三脑室。可替代地直接注入到第三脑室。 注意:这增强的特异性，但可能会导致降低的存活率（损坏的高风险）。 </li><li>通过应用专门钳型铂电极上的电压（间隔周期长度为50毫秒，间歇暂停950毫秒）。 <ol><li>使用一个0.5或3mm电极。 </li><li>使用电压范围为33〜35 V. </li><li>将电极的中心仅后路耳原基。 </li><li>以从25-40°染丘脑使用角度，以转染下丘脑90-180°（图2D）。 </li></ol></li><li>执行后电/术后护理作为DEScribed在1.4节。牺牲小鼠并按照第3条所述电（E18）后分析胚胎6天。 </li></ol></li></ol> 3.灌注固定<ol><li>准备工作<ol><li>温暖的4％多聚甲醛（PFA）至37℃。 </li><li>填在50ml的注射器用PBS（4℃）和第二注射器用温热的PFA。避免气泡。 </li><li>注射器连接到一个三路活塞。第三端连接到翼输液器（蝴蝶）。 </li><li>冲洗翅输液器，用PBS。 </li></ol></li><li>灌注<ol><li>深麻醉小鼠（怀孕或产后转）与盐酸氯胺酮皮下应用（60毫克/千克）和赛拉嗪（7.5毫克/千克）。 </li><li>经确认为无反应脚趾捏了手术耐受阶段。 </li><li>打开腹腔只是肋骨之下。 </li><li>小心切割膜片。 </li><li>小心地通过一个位于E剖开胸腔ACH网站到锁骨。 </li><li>将蝴蝶的尖端插入左侧心脏室（从心脏的顶点开始）。 </li><li>切开左心耳。 </li><li>慢慢灌注小鼠用PBS（约10毫升）中。 </li><li>切换活塞三路。 </li><li>灌注鼠标5-10毫升4％的煤灰。 </li><li>用剪刀斩首鼠标和解剖大脑开始在枕骨大孔。由于此前公布的16下进行清扫。 </li><li> Postfixate大脑对于在4％PFA 2天。保持大脑在4℃下在黑暗中。 </li></ol></li></ol> 4.免疫组化<ol><li>产生70微米的部分（例如，用一个vibratome）。 </li><li>可选:增强荧光抗体染色。 <ol><li>阻塞在RT 1小时的部分（0.1-0.2％的Triton X-100，5％正常山羊血清） </li><li>孵育O / N与合适的抗体（抗GFP，1:1000） </li><li>洗3-5倍用0.1M磷酸盐缓冲液中。 </li><li>孵育3-5小时用适当的荧光缀合的第二抗体（的Alexa Fluor 488缀合的抗兔IgG抗体，1:1000）。 </li><li>择:（在0.1M磷酸盐缓冲液的DAPI，0.2微克/毫升）染液4-6-diaminodino -2-苯基1分钟。 </li><li>用适当的荧光显微镜分析荧光。 </li></ol></li></ol>

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The authors have nothing to disclose.

This protocol describes in detail how to transfect 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 of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

自2001年第一次描述由三个独立的小组1-3 I N 子宫内的电已成为在啮齿动物中枢神经系统的分析基因表达的一种广泛使用的标准工具。相比一代基因敲除小鼠，这是，尽管不断提高技术，还有时间和金钱消耗，对在子宫内电由于其简单的呼吁。 所以 ，在子宫内的电实现了快速，高效的GAIN-和丧失功能的研究4。 转染脑区，包含带负电荷的质粒的溶液注入到心室。在电脉冲，带负电荷的DNA迁移朝向正极，因此，转染的区域可以通过改变阳极的位置简单地选择。它经常被显示，对中枢神经系统的许多区域可たrgeted 3,5-8。例如，最近的研究表明，海马，梨状皮层或纹状体9-11的特定转染。然而，关于适当的位置的信息往往只是很少标准化，并不总是容易转移到不同的小鼠品系。 某些萌芽阶段的转染是远离琐碎。当选择在子宫内的电的集式为特定的许多影响因素必须考虑。首先，以最佳转染各自胚胎阶段，知识适当的电压是必要的。高电压降低存活率，而低电压降低转染效率2,3,12。另外，电极桨的大小起着至关重要的作用，因为采用太大的效果降低特异性或可导致死亡是由于心脏节律4,12,13亲情电极桨。该应用电压的大小和电极桨的位置是要考虑的最重要的特征，但也有影响电穿孔的结果，等的DNA的溶液的施加量进一步的因素。 我们已经制定了详细的协议，它使C57BL / 6小鼠12的各个脑区的快速，高效的转染。在此要使用的协议有关的电压的详细信息，并设置在电极焊盘以增强特异性的大小。此外，关于心室信息要与用于质粒溶液的量和电极的位置的建议被供给填充的沿。在地图，这些位置的进一步可视化的详细位置信息的指示使简单的具体和有效的 retrosplenial皮层，运动皮层，躯体感觉皮层，梨状皮质，T的子宫内的电他CORNU ammonis 1-3，齿状回，纹状体，外侧隔核，丘脑和下丘脑。

<table><tbody><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Fast Green</td><td>Roth</td><td>0301.1</td><td></td></tr><tr><td>pCAGGS</td><td>Addgene</td><td></td><td></td></tr><tr><td>borosilicate glass capillaries (0.8-0.9 mm diameter)</td><td>Wold Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott</td><td>PZN 4831850</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>approval number: 400684.00.00</td><td></td></tr><tr><td>eye ointment (Bepanthen Augen und Nasensalbe)</td><td>Bayer&amp;nbsp;</td><td>PZN 01578681</td><td></td></tr><tr><td>0.9% benzyl alcohol 0.9% saline solution</td><td>Pharmacy&lt;br/&gt; of the University Medical Center Mainz</td><td></td><td></td></tr><tr><td>gauze (ES-Kompressen)</td><td>Hartmann</td><td>407835</td><td></td></tr><tr><td>sterile 5-0 Perma-Hand Silk Suture</td><td>Ethicon Johnson &amp;amp; Johnson</td><td>K890H</td><td></td></tr><tr><td>ring forceps 1/ 1.5 mm</td><td>Fine Science Tools</td><td>11101-09</td><td></td></tr><tr><td>ring forceps 4.8/ 6 mm</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>ring forceps 2.2/ 3 mm</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Adson Forceps-Serrated Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Mayo-Stille Scissors-Straight 15 cm</td><td>Fine Science Tools</td><td>14012-15</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Elektroporator CUY21 SC&amp;nbsp;</td><td>Nepa Gene Co.</td><td></td><td></td></tr><tr><td>FST 250 Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microgrinder EG-44</td><td>Narishige</td><td></td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Platinum electrodes 650P 0.5 mm</td><td>Nepagene</td><td>CUY650P0.5</td><td></td></tr><tr><td>Platinum electrodes 650P 3 mm</td><td>Nepagene</td><td>CUY650P3</td><td></td></tr><tr><td>Platinum electrodes 650P 5 mm</td><td>Nepagene</td><td>CUY650P5</td><td></td></tr><tr><td>Platinum electrodes 650P 10 mm</td><td>Nepagene</td><td>CUY650P10</td><td></td></tr><tr><td>Anesthesia system</td><td>Rothacher-Medical GmbH</td><td>CV-30511-3 Vapor 19.3</td><td></td></tr><tr><td>Heating plate</td><td>Rothacher-Medical GmbH</td><td>HP-1M</td><td></td></tr><tr><td>Temperature Controller 220V AC</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td></td></tr></tbody></table>

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.

图2 显示了发展成为retrosplenial皮层区域的子宫内的电示例，具体的，运动皮层，躯体感觉皮层，梨状皮质，大角ammonis 1-3，齿状回，纹状体，外侧隔核，丘脑和下丘脑。该转染的结果示旁边的推荐角度（ 图2）。为更好地可视化在体内角度的电极（0.5毫米）的位置也示于胚胎（图5，胚胎在子宫内 ，胚胎在羊膜囊，解剖胚胎）。为了...

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

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 ...

cortex-hippocampus-thalamus-hypothalamus-lateral-septal-nucleus

Research

JoVE Journal

Neuroscience

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

27.9K 次观看.  University Medical Center, JGU Mainz. 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.

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

科研

发育生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

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

生物学

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 ...

神经科学

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

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 ...

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, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

双电穿孔，以定时和空间分离的细胞群

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

通过子宫电位对脑神经祖细胞进行神经祖细胞的振动定位

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用

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