Name: ex utero electroporation and organotypic slice culture of mouse hippocampal tissue
Uploaded: 2015-03-04T15:00:00.000+00:00
Duration: 9 min 17 s
Description: Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

This work was supported by grants from the Deutsche Forschungsgemeinschaft to SB (BR-2215; SFB 497/A9).

注:所有的动物进行了实验，按照德国法律，并批准了在图宾根的政府部门。 1.准备微量移液器，解决方案和膜的<ol><li>微量移液器的制备<ol><li>使用微量拉马与以下程序拉玻璃微:热量:540，拉:125，速度:20和延迟:140针长度达5.5公分。 </li><li>使用microgrinder斜角针来获得4mm的合适的喷嘴尺寸。保存在盒子或15厘米培养皿针，以防止损坏的提示。 </li></ol></li><li>溶液的制备<ol><li>质粒DNA溶液<ol><li>制备含有根据制造商的协议使用不含内毒素马克西-prep试剂盒的所需的cDNA构建体的质粒DNA。 </li><li>调整质粒DNA溶液至3微克/微升的最终浓度（不GFP穗载体）或4微克/μL（含1微克GFP秒杀载体）中的内毒素释放含固绿（终浓度为0.05％）的Tris-EDTA缓冲。 </li></ol></li><li>层粘连蛋白原液<ol><li>溶解1毫克的层粘连蛋白在无菌水中，以1毫升的最终体积。制备100微升等分试样并储存在-80℃。 </li></ol></li><li>聚L-赖氨酸原液<ol><li>溶解在50ml无菌水中50毫克聚L-赖氨酸，以1毫克/毫升的最终浓度。准备1ml等份并储存在-20℃。 </li></ol></li><li>完成Hank氏平衡盐溶液（HBSS完成） <ol><li>准备完成的HBSS通过将100毫升10×HBSS，2.5毫升的1M HEPES缓冲液（pH7.4）中，加入30ml的1M D-葡萄糖，加入10ml的100mM的氯化钙 ，将10毫升的100mM的硫酸镁 ，和4毫升的1M的NaHCO 3。加无菌水至1升，并储存在4℃。 注:高压灭菌的所有解决方案期望的1M HEPES缓冲液和1M D-葡萄糖，这是FIL器消毒。 </li></ol></li><li>切片培养基<ol><li>通过加入35毫升基础培养基鹰，12.9毫升完整的HBSS（1.2.4），1.35毫升的1M D-葡萄糖，250微升200mM的L-谷氨酰胺，和500微升制备切片培养基青霉素 - 链霉素，以获得50 ml的终体积。添加马血清至5％的终浓度，并储存在4℃。 </li></ol></li><li>低熔点（LMP）琼脂糖<ol><li>通过加入2克LMP琼脂糖至50ml完整的HBSS（1.2.4），随后加热在微波中以高功率1-2分钟制备4％LMP琼脂糖溶液。保持此解决方案在水浴在37 - 39℃。存储所述溶液在4℃和重用。 </li></ol></li><li>多聚甲醛溶液<ol><li>在通风橱中通过加入4g多聚甲醛至100毫升的1×PBS中制备的4％多聚甲醛（PFA）的解决方案。加热该溶液至60℃并加入几滴1N的NaOH直至溶液变成C李尔。 </li></ol></li><li>通透的解决方案<ol><li>溶解9克的BSA在300毫升的1×PBS中含有0.3％Trition X-100和储存在4℃。加10％的叠氮化钠于长期贮存。 </li></ol></li></ol></li><li>插入膜涂层<ol><li>稀释的层粘连蛋白原液（1.2.2）和在无菌水中聚L-赖氨酸原液（1.2.3）一个等份等分试样1到12 ml的终体积。 </li><li>放置膜插入到6孔板每孔含有2毫升无菌水中。加入1毫升的涂料溶液在膜的顶部，并在5％CO 2的培养箱中孵育O / N在37℃。 </li><li>温育后洗涤膜插入三次用1ml无菌水，干燥。在干燥6孔板用在同一天或商店涂布的膜插入物在4℃下长达四个星期。 </li></ol></li></ol> 2. DNA注入和E15.5的电穿孔和E18.5胚胎<ol><li&gt;通过将其放置到饱和，用5％异氟醚，并且连接到一个蒸发器的麻醉箱麻醉交配雌性小鼠一次。流通的异氟烷和氧气以1升/分钟的速率。保持在箱子的动物为2-4分钟，或直至无意识其由鼠标的爪子之间夹持测试。 </li><li>通过对胚胎天颈椎脱位（E）15.5 18.5安乐死无意识鼠标。解剖含13胚胎在子宫并将其放入含有15-20毫升冷HBSS完整的培养皿。 注意:从这时开始，保持胚胎和组织上的冰块。 </li><li>用剪刀从子宫角和地点每个胚胎中分离到含有完整的冷HBSS第二个培养皿中。 </li><li>在解剖显微镜下，切断子宫肌壁，用一对细镊子（＃55）和剪刀的胎盘。仔细释放从卵黄囊胚胎。 </li><li>使用一对波恩剪刀来decapitatE中的胚胎刚刚前肢在60°角以上。如果实验需要的胚胎基因分型，收集的基因组DNA分离的组织样品（一小块尾巴）。 </li><li>头转移到一个干净，干燥的培养皿。因为头部被断头在一个60°角，当置于背侧的头应该倾斜到一侧。 </li><li>小心地注入了一针半球接近前囟门的中间（ 图1A，B）。通过步进用30磅的压力为每脉冲10-15毫秒的持续时间的picospritzerⅢ的踏板上，施加5-8脉冲注入约2-3微升（在3或4微克/微升）DNA溶液。脉冲的持续时间和数量取决于针开口与较小的开口，需要更多的时间的直径。每个脉冲之间的间隔等于1秒。 </li><li>前放置电极，应用几滴完整的HBSS对电磁制动的头哟。放置电极以这样一种方式，"负"终端是在同一侧的注入脑室和对胚胎的头部（ 图1C，D） ​​的耳下方注入脑室的相对侧的"正面"电极。应用5脉冲50 V. <ol><li>使用3毫米电极对于E 15.5 5毫米的电极对于E 18.5。 </li></ol></li></ol> 3.解剖的大脑<ol><li>在电穿孔后，撕下从头部有一对细镊子的帮助下皮肤。利用一对弹簧剪刀作一小切口在小脑的中部在颅骨的中线。 </li><li>将弹簧剪刀插入切口切开纵向沿矢状缝。剥离头骨，并通过使用细镊子取下从头骨的大脑。转让全脑入15-20毫升冷HBSS完整的解决方案。 </li><li>与此同时，倒入4％LMP琼脂糖，保持在37-39℃的水浴中，进一剥离式模具。 </li><li>取脑出完整的HBSS通过小铲子铲，并通过使用细棉纸或的Kimwipes排出多余的HBSS。 </li><li>轻轻地放在全脑入琼脂糖，并调整其用细针的位置。保持在冰上的模具，直到琼脂糖是固化和块被分段（对于冠状切片嗅球指向向上）。 </li></ol> 4.的vibratome切片和切片培养<ol><li>修剪LMP琼脂糖块和胶水他们使用"超级胶"的样品台。胶后的干式转印试样阶段到的vibratome的缓冲盘和填充用冰冷的HBSS完成直到该块被浸在溶液中。 注:切片前消毒所有的仪器和设备表面用70％乙醇。 </li><li>使用新的刀片准备250微米厚的切片的vibratome作为紧随其后。 <ol><li>修剪块无线个以下设置;频率 - 60赫兹，振幅 - 0.7微米，速度16〜18毫米/秒。切割含有与低速（9毫米/秒）的上述设置的期望的组织的部分。开始从后脑切片，收集5-7部分来自大脑。 </li><li>该部分转移到含有5ml冰冷的HBSS完成与弯曲锅铲的帮助，并保持在冰上，直到所有的部分都收集（ 图1E）干净的6孔培养皿。 </li></ol></li><li>滋润在用100μl完全的HB​​SS的横时尚膜放置部分上的膜之前，以促进各部分的取向。 </li><li>传送用的弯曲刮铲的部分到膜（拿起部的角与钳和拉入刮刀，然后使用镊子的部分推到膜上）。放置最多五个部分上的一个膜，并通过使用镊子安排（ 图1F）。不相互重叠的部分。 <ol><li>取过量的HBSS断使用移液管的膜。此处所使用的具体膜附连到框架，并插入到组织培养板，其允许组织是在接触，但不包括在介质。 </li></ol></li><li>将膜插入到含有1.8毫升切片培养基（1.2.5）（ 图1G，H）的一个6孔板中。孵化培养皿在37℃，5％CO 2的11格或14格。改变一半的中等（0.9毫升）每隔一天。 注:在此阶段，加入试剂像溴脱氧尿苷（BrdU的;最终浓度10μM）用于标记增殖细胞的培养基的第20小时的培养时间。 </li></ol> 5.固定的章节其次是免疫荧光染色<ol><li>使用干净和锋利的手术刀刀片切割和修剪视的取向膜的章节。 </li><li>沿着与膜传送部分以含有1ml 4％PFA（1.2.7）的24孔板。孵育切片1小时，在室温随后3次洗涤用1×PBS，每次15分钟。孵育切片O / N与透液在4℃下温和搅拌。 </li><li>翌日，孵育适当的一抗的章节中，稀释于透化溶液，O / N或48小时，在4℃下温和搅拌。 </li><li>洗第3次15分钟，用1×PBS孵育O / N在4℃下与稀释在透化溶液中的合适的二级抗体。 </li><li>与第二抗体的温育后，用1×PBS洗涤一次的部分15分钟，然后用DAPI染色10分钟。 </li><li>洗切片3次用1×PBS，每次15分钟，并转移到显微镜载玻片上。添加ImmunoMount，轻轻放在段顶部盖玻片。干幻灯片O / N在4°C和SE人与指甲油。 注意:保持幻灯片始终在4℃。 <ol><li>分析切片文化共聚焦显微镜（ 图1I）。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

海马在学习和记忆的重要功能。齿状回也是在那里发生神经不仅在发展，而且在整个成年期2脑区之一。产后和成人海马神经发生的收益在涉及许多共同的因素类似的方式。限定这些因素的调节机制将在理解神经变性疾病这又会导致新的治疗和预防措施非常有益的。要获得此信息的一个需要一个系统来操纵单个细胞，并就证明了前子宫内电后器官切片文化观察他们在家乡的环境。 <p class="...

海马起着记忆和学习中起重要作用，以及情感行为。其中一个主要功能包括整合的短期记忆转化为长期记忆，这需要神经系统的可塑性高的。海马的齿状回充当用于输入信息的主网关，也是两个脑区与正在进行的神经发生1整个成年期1,2。海马结构的发育过程中胚胎发育后期，尤其在最初3〜4周，产后3时。在齿状的早期发展回中需要设一个干细胞库是用于产后以及成年神经4。显影神经元产后以及成年神经发生过程中通过各种阶段，从干细胞通过祖细胞向未成熟和最后的成熟神经元的几个阶段。在神经发生中的表达的不同阶段特定的基因是必需的，以允许成熟和集成新的神经元的进海马电路5,6。 用小鼠遗传学和免疫表型分析，以及允许定义许多这些基因的表达模式和功能分子方法。在另外的微阵列分析，以及染色质免疫沉淀（ChIP）提供关于潜在的直接和间接的靶基因7,8-信息。然而，仍然有关于海马发育的调控机制许多悬而未决的问题，在齿状回尤其是发展。通过向下或向上调节的兴趣和/或它的靶基因和发育过程中按照它们的命运的基因的进一步了解如何特定基因调节的系统是必需的，允许小数量的单元的操作， 在子宫内电的shRNA，利息或Cre重组酶毒素重组基因的cDNA的本身提供了这样一种工具。以确保所希望的DNA或小RNA表达质粒应当用于电穿孔的存在。这种做法是非常成功实施的研究皮质发育9,10，但它是一个更具挑战性的方法检查齿状回中的发展，由于海马结构的更深层次的脑的位置。 前子宫内电后器官切片文化是一种方法来解决这个问题11,12。与此相反，以在子宫内电不是整个胚胎但只有头部被用于允许因此放置电极以更有利的方式直接向海马和齿状回shRNA的/ DNA。我们的团队成功地采用前子宫内电齿状回中的发展过程中8，研究转录因子BCL11B的作用。 BCL11B在齿状回中的发展为r的双重角色egulating祖细胞的增殖和分化的证明了免疫组化。进一步定义了一种机制，BCL11B参与这些过程中，Polleux组11,12的协议进行了调整，以研究在协议部分下面描述的齿状回。在第一种方法的问题是解决BCL11B是否自主调节神经细胞分化的细胞。第二种方法检查桥粒，BCL11B的直接靶基因是否足以拯救BCL11B表型。

<table><tbody><tr><td>Flaming/ Brown Micropipette Puller</td><td>Sutter Instruments Company (USA)</td><td>P-97</td><td></td></tr><tr><td>Fine Glass Pipettes</td><td>Warner Instruments</td><td>G100F-4</td><td></td></tr><tr><td>Microgrinder</td><td>Narishige, Japan</td><td>EG-44</td><td></td></tr><tr><td>Anesthetic Bracket unit</td><td>Harvard Apparatus</td><td>PY2 34-0412</td><td></td></tr><tr><td>Halovet Vaporizer</td><td>Harvard Apparatus</td><td>PY2 34-0398</td><td></td></tr><tr><td>Fluovac System</td><td>Harvard Apparatus</td><td>PY2 34-0387</td><td></td></tr><tr><td>IMS Fluosorber</td><td>Harvard Apparatus</td><td>PY2 34-0415</td><td></td></tr><tr><td>Anesthetizing Chamber</td><td>Harvard Apparatus</td><td>PY2 34-0460</td><td></td></tr><tr><td>Electroporator</td><td>BEX Company</td><td>CUY21 EDIT</td><td></td></tr><tr><td>Tweezers with disk electrodes</td><td>BEX Company</td><td>LF650P3</td><td>3 mm electrodes for E15.5</td></tr><tr><td>Tweezers with disk electrodes</td><td>BEX Company</td><td>LF650P5</td><td>5 mm electrodes for E18.5</td></tr><tr><td>Picospritzer III</td><td>Parker Hannifin Corporation</td><td>P/N 052-0500-900</td><td></td></tr><tr><td>HM 650 V Vibrating Blade Microtome, 230 V</td><td>Thermo Scientific</td><td>920120</td><td></td></tr><tr><td>Dissection Microscope</td><td>Carl Zeiss Microscopy Gmbh</td><td>Stemi SV8</td><td></td></tr><tr><td>Inverted Microscope</td><td>Leica</td><td>Leica DM IL LED</td><td></td></tr><tr><td>Confocal Microscope</td><td>Leica</td><td>Sp5II</td><td></td></tr><tr><td>6 well dish</td><td>BD Falcon</td><td>#353502</td><td></td></tr><tr><td>6 well dish</td><td>CELLSTAR</td><td>#657160</td><td></td></tr><tr><td>Tissue culture inserts</td><td>BD Falcon</td><td>#353090</td><td></td></tr><tr><td>Fast Green</td><td>Sigma</td><td>F7252</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>#L2020</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>#P5899</td><td></td></tr><tr><td>Spring scissors</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Forceps</td><td>Dumont #55</td><td>11255-20 Inox</td><td></td></tr><tr><td>HBSS 10x</td><td>Life Technology</td><td>14180-046</td><td></td></tr><tr><td>BME</td><td>Life Technology</td><td>41010-26</td><td></td></tr></tbody></table>

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.

转录因子BCL11B消融导致的祖细胞增殖并导致降低的齿状回的大小和细胞数目的神经元分化的损害。此外突变的神经元无法融入海马电路引起的学习记忆障碍8。要回答有关BCL11B在这些过程中的前子宫内电被采用的监管机制（S）的问题。 解决这个问题是否BCL11B细胞自主调节神经细胞分化，分 ​​别由GFP-Cre重组酶结构或单独GFP 11的前子宫内电...

Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

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 ...

ex-utero-electroporation-organotypic-slice-culture-mouse-hippocampal

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

9.5K 次观看.  University of Ulm. 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.

视频: Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

前子宫穿孔和整个半球的外植体早期皮质发育的研究：一个简单的实验方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

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 ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

小鼠河马兴奋和类特异性抑制神经元的不同组织型切片培养的单细胞电位

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Preparation of Organotypic Slice Cultures from a Rat Pup&#39;s Hippocampus

Source: Mosa, A. J. et al., <a href="https://app.jove.com/v/52353">Organotypic Slice Cultures for Studies of Postnatal Neurogenesis.</a> J. Vis. Exp. (2015).
This video demonstrates a method for preparing organotypic hippocampal slice cultures from the brain of a rat pup. It outlines the steps involved in the dissection of the hippocampus, slicing, and culture conditions required to promote cell viability and growth in the hippocampal slices, ultimately generating organotypic slice cultures.

从大鼠幼犬的海马体制备器官型切片培养物

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

小鼠海马组织的前子宫内电和器官切片文化

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