Name: ex vivo culture of chick cerebellar slices and spatially targeted electroporation of granule cell precursors
Uploaded: 2015-12-14T15:00:00.000+00:00
Duration: 10 min 2 s
Description: Watch this Scientific Journal Video about Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors at JoVE.com

在这篇文章中介绍的方法源于由BBSRC BB /资助的工作I021507 / 1（TB，RJTW）和MRC博士助学金（MH）。

注:所有实验均用按照国王学院，英国伦敦和英国内政部的动物护理准则进行的。 1.解剖E14小脑<ol><li>在38℃孵育棕色受精母鸡的蛋胚胎第14天。 </li><li>使用蛋剪刀斩首鸡胚卵内并取出头到含有冰冷的PBS（图1A）培养皿。 </li><li>使用标准镊子，使切口每只眼睛后面所有的方式，通过组织，消除眼睛和上颚。让所有的方式，通过咽去除下颌（ 图1B），第二个切口。 </li><li>使用标准镊子，通过剥离它扔掉（ 图1C）从头骨的表面上的所有皮肤，去除额骨和顶骨，揭示大脑。 </li><li>从腹侧方面​​，除去咽软骨和辅助间充质。 </li><li>从背侧，小心LY去除间充质背到后脑，注意不要损坏软，以及独立的整个大脑（ 图1D）。 </li><li>一路经中脑和后脑和独立的后脑，包括小脑（ 图1E）之间的组织做切口。 </li><li>通过所有的方式，通过组织在小脑和后脑的翼片的两侧路口做切口，去除整个小脑，注意保持PIA的整个解剖的完整性（ 图1F）。取出形成脉络丛（ 图1G）。 </li><li>移动解剖小脑入冰冷的HBSS中。 </li></ol> E14小脑2.切片培养<ol><li>整个小脑传送到使用刮刀或3 ml的巴斯德吸管与尖端一个组织斩波的无菌平台切去加宽孔。用吸管去除多余的液体。 </li><li>切在T整个产品小脑他使​​用该组织的切刀设定具有切割速度在最大的50％所需的取向，在300微米的厚度。组织的完整性是最容易被保存在矢状切面;然而，取向也是细胞分析的重要考虑因素（浦肯野细胞树突矢状对齐，而颗粒细胞轴突横向运行，垂直于浦肯野细胞树突的平面）。 </li><li>使用3mL的巴斯德吸管，覆盖在冰切片小脑冷的HBSS。 </li><li>转让于HBSS切片到含有冰冷的新鲜的HBSS使用3 ml的巴斯德吸管切头一个60毫米的培养皿。 </li><li>在解剖显微镜下照射光纤光源，确保使用钟表制造商镊子各个切片的分离。识别片被电穿孔，根据他们组织的完整性和内 - 外的位置。 注:从胚胎通过切片孵化培养基中的每个夹层大约需要10-20分钟取决于UPOñ经验。进行解剖一次，以确保小脑花费最短时间斩首和体外培养的。 </li></ol> 3.电穿孔切片<ol><li>通过电穿孔的阳极固定到60毫米的培养皿用绝缘带的基部构造电穿孔室。添加约1 ml的HBSS中，以覆盖电极。 </li><li>上放置覆盖在HBSS电极顶部上的0.4微米的培养插管。允许培养插管休息的电极确保总有插入件与电极之间的接触。 注意:在此设置中，培养插管的切片将休息的溶液的表面上，保持电路，但允许在阴极的空间定位。 </li><li>传输标识切片（最多五个每培养插入）到使用3 ml的巴斯德吸管与切口尖端培养插管。独立，并允许定居到文化插入在sagitt人的方向。 </li><li>使用移液管，除去过量的HBSS以使切片不再溶液沐浴。 </li><li>使用P10移液管尖，吸管的DNA（在1微克/微升的浓度）用20％的快绿放在切片的目标区域的表面稀释。 20％的快速绿色确保粘稠的DNA溶液，防止DNA望而却步广泛散布。添加约5微升的DNA /快绿溶液到每个切片（图2B）。 </li><li>放置在阴极上所需的目标组织电穿孔和用3×10伏，10毫秒持续时间的脉冲。避免与组织中的阴极直接接触。相反，采取的液体的表面张力的优点维持电导（放置阴极尽可能靠近组织尽可能没有实际接触的组织）。 </li><li>根据需要重复DNA递送和电到EGL的多个区域每个人的小脑切片。 </li><li>在电穿孔完成后，转移培养樱雪室温至30毫米的培养皿。 </li><li>向每个培养，加入1毫升预热的培养基（基础培养基Eagle，0.5％（重量/体积）D - （+） - 葡萄糖，1％B27添加剂，2mM的L-谷氨酰胺，100U / ml青霉素， 100微克/ ml链霉素）的培养插管，使得培养插管是在与介质接触，但片没有在它沐浴下方。 </li><li>孵育培养物，在37℃/ 6％CO 2的最多3天。更换所有培养基中，每24小时用新鲜预热培养基。 </li><li>继文化，固定片（在4℃或O / N）上培养插入1小时在4％多聚甲醛在新的30毫米的菜，没有文化传媒。 </li></ol> 4.成像小脑切片<ol><li>以下定影，洗涤切片3次，每次5分钟，于PBS中。 </li><li>用剃刀刀片，通过切割切片周围并除去切片并插入它粘附到的区域解剖从培养插入物的每个电穿孔和培养的小脑切片。做不试图从培养插入物表面上除去切片。 </li><li>下一个盖玻片小心在大约1毫升含有DAPI（如果需要的话）的安装平台的安装片不引入气泡，并使用激光扫描共聚焦显微镜图像。 注意:成像参数可根据电穿孔将构建而改变，在个人用户的判断。 </li></ol>

The authors have nothing to disclose.

这里所报告的协议描述了解剖，电穿孔和从小鸡培养的胚胎第14天小脑切片的方法。该协议使电的靶向东瀛小灶地区，包括个别的小脑叶片孤立的目标。它使遗传分析和成像以高分辨率和便利性，并以较低的成本相比，在啮齿类动物中43-47建立的技术。这样的分析是目前不可能在体内 ，由于延长的发育时间段，在小鼠EGL特异性遗传靶向的可能性很少，和的菱形唇与EGL之间的分子机制...

小脑坐在后脑的前端，并负责感觉和运动处理中的成熟大脑的整合以及调节更高的认知过程1。在哺乳动物和鸟类，它具有一个精心形态并被大量面理化，的祖细胞的广泛过境扩增产物产生超过一半在成人脑的神经元的发育过程中。小脑一直研究的课题的神经生物学家了几个世纪，在分子的时代也同样收到显著的关注。这不仅涉及其固有的吸引生物学，而且这样的事实，它在很大程度上与人类疾病包括发育的遗传性疾病如孤独症谱系障碍2，最突出的是小脑癌，髓母细胞瘤3，这是最普遍的儿童脑瘤。重要的是，它是内的wh的优良模型系统非物质文化遗产研究的命运分配和神经大脑发育过程中4。近年来，它也被确立为大脑发育的比较研究模型系统，由于对整个可见的脊椎动物系统发育5-10小脑形态的巨大差异。 小脑从菱1在后脑11背侧半开发和发育是由两个主要祖种群，菱形唇缘和脑室区的。周围的后脑的神经上皮的背部区域的菱形唇延伸与屋顶板的边界。它是小脑12-14的谷氨酸能神经元的兴奋性的发源地。脑室区产生了抑制GABA能神经元的小脑，最突出的是大浦肯野神经元14,15。后来在发展（约13.5小鼠胚胎天; E6的小鸡16），谷氨酸培罗成itors从菱形唇沿切线方向迁移，形成祖细胞的软脑膜层:所谓的外部颗粒层（EGL）二级祖区。正是这层经过广泛的过境放大，导致颗粒神经元的数量巨大，在成熟大脑中发现。 增殖在EGL早已被链接到子软脑膜位置而导致的切向迁移从菱形唇17，与交换机细胞周期出口和祖细胞的神经元分化正在与他们的出口从外EGL层到中间关联EGL 18。有丝分裂后的颗粒细胞的中间-侧向轴线广泛切线迁移发生在中间和内部的EGL 19，最终径向迁移进入成熟小脑皮层的内颗粒层之前。从菱形唇在小脑表面细胞的迁移是依赖于CXCL12信号从软20-22 </sup&gt;和颗粒细胞表达CXCL12受体CXCR4。他们的切向迁移是这样回忆那新皮质切向迁移抑制中间神经元群体23-25。有趣的是，电子显微镜研究17表明，EGL细胞增殖形态保持软脑膜接触，连接细胞的行为具有增殖能力的让人联想到哺乳动物的大脑皮层26的基底祖细胞的方式。这反映在EGL的前述分层成三个子层是由不同的胞外环境，并在那里颗粒前体具有不同的基因表达签名18限定 。 在oEGL祖细胞增殖发生克隆大小正态分布使得当祖单独遗传标记在小鼠胚胎发育的末端，它们产生的有丝分裂后250-500克的中值平均ranule神经27,28。扩散是依赖于有丝分裂SHH信号来自底层的浦肯野神经元29-32。向SHH作出反应的能力已被证明是完全依赖于转录因子ATOH1的细胞中自主表达， 在体外33 和体内34,35。同样地，细胞周期出口和分化已被证明是取决于下游转录因子NEUROD1 36，这很可能ATOH1 37的直接阻遏的表达。 尽管这些进展，并且在解密的细胞周期出口38-42的细胞生物学的基础相当大的进步，根本的分子机制（S）所依据决定退出细胞周期和从祖过渡到微分神经元，并且在内侧的EGL关联的有丝分裂后的切向迁移以及后来的开关以放射状迁移，仍然不完全理解。这是为了由于EGL的实验棘手的很大程度:它是迟显影，而且很难基因靶向，因为许多相同的神经性分子，可以在颗粒的前体的寿命的关键早些时候在菱形唇。为了解决这个问题，许多作者已经开发出在体内和体外电作为一种方法，目标在啮齿类动物中43-48产后小脑。在这里，我们率先使用体外电穿孔在小鸡研究EGL，它代表在成本和便利方面相当大的优势。我们的电穿孔和体外切片鸡小脑组织培养的方法使用组织解剖从胚胎第14天的雏鸡在EGL扩散的高峰期。这种方法允许菱形唇的EGL的遗传靶向独立地，将设置了阶段为从颗粒的过渡的遗传解剖祖在有丝分裂后小脑颗粒神经元。

<table><tbody><tr><td>McIlwain tissue chopper</td><td>Mickle Laboratory Engineering Ltd</td><td></td><td>Cut at 300 &amp;mu;m for best results.</td></tr><tr><td>Basal Medium Eagle (Gibco)</td><td>Life Technologies</td><td>41010-026</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>0.4 &amp;mu;m culture insert</td><td>Millipore</td><td>PICM0RG50</td><td></td></tr><tr><td>TSS20 Ovodyne electroporator&amp;nbsp;</td><td>Intracel</td><td>01-916-02</td><td>Use 3 x 10 V, 10 msec pulses for electroporation.</td></tr></tbody></table>

ex vivo culture of chick cerebellar slices and spatially targeted electroporation of granule cell precursors

小脑外部颗粒层（EGL）是最大的中转放大在发育中的大脑的网站，并为研究神经元的增殖和分化一个很好的典范。另外，其增殖能力的进化改进一直负责小脑大小在羊膜急剧扩张，使得小脑为脊椎动物脑的EVO发育生物学研究的优良模型。东瀛的组成细胞，小脑颗粒祖细胞，也代表来源的显著细胞的髓母细胞瘤，最常见的小儿神经肿瘤。以下中转扩增，颗粒的前体的径向迁移进入其中它们代表最大的神经元群中的成熟哺乳动物脑小脑的内部颗粒层。在小鸡，东瀛增殖高峰出现对妊娠的第二周结束。为了目标遗传修饰该层在泛滥的高峰期，我们已经制定了通过小脑切片的离体电穿孔的基因操纵从胚胎第14天鸡胚的方法。这种方法概括体内颗粒神经元发展的几个重要方面，并会生成一个透彻的了解小脑颗粒细胞的增殖和分化是有用的，因而小脑发育，进化和疾病。

本节说明可使用切片电和小脑的文化胚胎第14天的小鸡得到的结果的例子。小脑的解剖示于图1和电穿孔室设置示于图2，我们表明，这是可能的电穿孔，并成功地培养小脑切片，仍保持其结构和细胞形态在体外 （图3A）。目标电穿孔到个别的Folia容易实现（图3B）。我们成功地电穿孔的一些?...

Watch this Scientific Journal Video about Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors at JoVE.com

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

小脑外部颗粒层是最大的过境扩增在显影脑的部位。在这里，我们提出了一个协议，以目标基因改造这个层扩散采用体外电击和小脑切片文化从胚胎第14天鸡胚的高峰。

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 ...

ex-vivo-culture-chick-cerebellar-slices-spatially-targeted

Research

JoVE Journal

Developmental Biology

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

9.2K 次观看.  King's College London. 小脑外部颗粒层是最大的过境扩增在显影脑的部位。在这里，我们提出了一个协议，以目标基因改造这个层扩散采用体外电击和小脑切片文化从胚胎第14天鸡胚的高峰。 

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

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

科研

生物学

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 ...

发育生物学

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

神经科学

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

器官细胞生存在器官细胞切片培养

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Source: Bovio, P., et al., <a href="https://app.jove.com/v/56631">Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup&#39;s cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

分离和培养小鼠小脑颗粒神经元祖细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations

	Preparations ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

Ex Vivo Electroporation of Chick Embryo Cerebellar Slices: A Method to Introduce Plasmid DNA Encoding Green Fluorescent Protein to Visualize Granular Cell Development

Source: Hanzel, M. et al.&#160;<a href="https://www.jove.com/video/53421" target="_blank">Ex Vivo&#160;Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.</a>&#160;J. Vis. Exp. (2015)
In this video, we perform electroporation of chick embryo cerebellum slices with reporter gene plasmids. The technique facilitates visualization of the development and migration of granule cell precursors to form granular layer.

鸡胚小脑切片的离体电穿孔:一种引入编码绿色荧光蛋白的质粒 DNA 以可视化颗粒细胞发育的方法

Source: Hanzel, M. et al.&#160;Ex Vivo&#160;Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.&#160;J. ...

癌症研究

Cancers of the Nervous System

In vitro studies

Obtaining Cerebellum Slices from an Embryonic Chick Brain

Source: Hanzel, M., et al. <a href="https://app.jove.com/v/53421">Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.</a> J. Vis. Exp. (2015).
The video demonstrates the preparation of cerebellum slices. It shows a detailed procedure for isolating the cerebellum from a chick embryo. The slices are then prepared from the dissected cerebellum using a tissue chopper for further experimental analysis.

从胚胎雏鸡脑中获取小脑切片

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

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

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产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

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

小脑颗粒神经元的遗传操作

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

器官细胞生存在器官细胞切片培养

利用体内出生后电穿孔研究小脑颗粒神经元形态和突触发育

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

分离和培养小鼠小脑颗粒神经元祖细胞

鸡胚小脑切片的离体电穿孔:一种引入编码绿色荧光蛋白的质粒 DNA 以可视化颗粒细胞发育的方法

从胚胎雏鸡脑中获取小脑切片

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

摘要

探索更多视频

此视频中的章节

产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

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

小脑颗粒神经元的遗传操作

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

器官细胞生存在器官细胞切片培养

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

分离和培养小鼠小脑颗粒神经元祖细胞

鸡胚小脑切片的离体电穿孔:一种引入编码绿色荧光蛋白的质粒 DNA 以可视化颗粒细胞发育的方法

从胚胎雏鸡脑中获取小脑切片

利用体内出生后电穿孔研究小脑颗粒神经元形态和突触发育