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 border="0" cellpadding="0" cellspacing="0" width="271">
	<tbody>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Name of Reagent/ Equipment</td>
			<td style="width:73px;">Company</td>
			<td style="width:63px;">Catalog Number</td>
			<td style="width:64px;">Comments/ Description</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Flaming/ Brown Micropipette Puller</td>
			<td style="width:73px;">Sutter Instruments Company (USA)</td>
			<td style="width:63px;">P-97</td>
			<td style="width:64px;"></td>
		</tr>
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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

9.4K 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

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

诱导蛋白缺失的通过<em&gt;在子宫内</em&gt;电穿孔来定义赤字的神经细胞迁移的转基因模型

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

科研

发育生物学

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

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 ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

发育小鼠垂体的解剖和冠状切片制备

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.