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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">McIlwain tissue chopper</td>
			<td style="width:71px;">Mickle Laboratory Engineering Ltd</td>
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			<td style="width:167px;">Cut at 300&#956;m for best results.</td>
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			<td height="63" style="height:63px;width:216px;">Basal Medium Eagle (Gibco)</td>
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			<td style="width:119px;">41010-026</td>
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			<td height="21" style="height:21px;width:216px;">L-glutamine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G7513</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4&#956;m culture insert</td>
			<td style="width:71px;">Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">TSS20 Ovodyne electroporator&#160;</td>
			<td style="width:71px;">Intracel</td>
			<td style="width:119px;">01-916-02</td>
			<td>Use 3x10v, 10ms pulses for electroporation.</td>
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</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

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 ...

科研

发育生物学

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

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 ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜生长培养测定和<em&gt;离体</em&gt;心外膜源性细胞迁移的评估

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

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 ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

啮齿类动物胎盘的体外灌注

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

成年斑马鱼端脑中疟原虫DNA在活体传递的电穿孔方法

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...