Name: rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord
Uploaded: 2014-11-01T14:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

CYIY is supported by FAPESP (2012/14421-5) and FMV is supported by a fellowship from FAPESP (2009/53695-0). We thank the DNA Technologies Core at University of California, Davis for preparation and sequencing of the RNA-Seq libraries used in this work, the Galaxy team for providing an excellent and free interface for high-throughput sequencing data analysis tools and Dr. Marianne Bronner for allowing us to film in her laboratory space.

1.电穿孔兴趣的HH12-13鸡胚胎的神经管在大毛的构建用荧光报道，优选在双顺反子转录物或融合于目的蛋白质与嵌入病毒2a中的肽11，以使快速识别与转染的令人满意的水平的个体。注意:典型的孵育时间为这个阶段是围绕48小时，在37.8℃。 <ol><li>除去3毫升白蛋白与耦合到18G的X 1 1/2针头的注射器后，打开一个小窗口中的鸡蛋。以提高胚胎的可视化，注入少量的墨汁在无菌林格氏胚胎1-4下方溶液以1:50稀释的。 </li><li>覆盖用无菌林格氏溶液中的胚胎后，注入含有2.5微克/微升通过神经管的后端的DNA直到填满一个的10mM的Tris-HCl，pH值8.2和0.1％食品染料（F，D＆amp; C）溶液到后脑区域。电穿孔，通过4毫米1沿侧翼区域分离位置的铂电极（直径0.5mm），并提供5个脉冲的50毫秒和20 V与他们4之间100毫秒的间隔。 </li><li>电穿孔后，孵化操纵胚胎48小时以上，直到他们达到的阶段HH23，并确定个人在日光灯立体呈现转高层次的侧翼区域。 注:孵育电穿孔后的持续时间取决于实验的目的。电穿孔质粒可以在2小时后2产生显著的表达水平。因此，如果兴趣在于发病早期基因的电穿孔后潜伏期可以减小。 </li></ol> 2.收获成功转染胚胎解剖和电穿孔脊髓减半注意:此过程持续2-3个小时，每10胚胎和产量1-2每个胚胎微克总RNA。神经管的解剖，需要精细动作，并把它应用到实验样品前，一些练习是必要的。 <ol><li>净化用RNase去污溶液或通过在150℃下烘烤4小时12金属清扫工具和制备DEPC处理的PBS中。只使用新的认证无RNA酶的塑料制品。 注:收获后，按照标准的护理，防止清扫时RNase污染是很重要的。 </li><li>收获用一双精致的解剖剪刀和过筛收集勺胚胎。保持在100毫米的培养皿中冷DEPC处理过的PBS胚胎。 </li><li>用一双细镊子，切出整个侧翼区（前肢芽和后肢芽的前极限的后限之间），这部分转移到含冷DEPC处理过的PBS一道新菜。 </li><li>用两个细玻璃针穿孔在三个不同的点于神经管和近轴中胚层之间的胚胎各背外侧边。 </li><li>引入两个玻璃针，在中央​​穿孔，慢慢移动中的一个远离沿着神经管前后轴。重复此步骤不是由所述第一运动达到该穿孔。用一双细镊子取出分离旁轴中胚层。 </li><li>把胚胎侧身，插入神经管和脊索之间的镊子的提示和他们在沿前后轴分开的两个结构相反的方向滑动。 注意:这也应该除去大部分的中胚层的仍然附着在神经管。 </li><li>如有必要，删除仍然受到拉动用镊子，同时轻轻握住神经管与另一镊子附加任何大块mesodermic组织。完成后，轻轻的神经管含冷DEPC处理过的PBS采用转移到另一道菜耦合无RNase玻璃巴斯德吸管向手动移液管泵，并保持在冰上，直到所有的管子都解剖。 </li><li>分化神经管成两半。对于此，插入一个封闭镊子在管腔的尖端和通过移动镊子出通过它打开屋顶板。在此之后，通过切割底板与镊子尖端分开两半。 </li><li>来自非电穿孔半部排序电下的荧光解剖显微镜和转移到1.5ml管中填充用冰冷的DEPC处理过的PBS中。如果需要的话，轻弹的微管轻轻地释放附着在塑料壁的任何组织，并促进管的沉淀。 </li><li>旋转100 XG为3秒，取出非常缓慢尽可能多的PBS尽可能与1毫升微量。做到这一点，而在寻找一个光源照射，通过微管监测如有胚胎物质被吸入到枪头;如果发生这种情况，慢慢地排出，并重复centrifuga化。 </li><li>加入70微升的每个神经管半RNA提取裂解液和轻弹试管混合内容。在室温下孵育5分钟，并大力旋涡。重复5分钟温育和涡旋两次以上。储存在-80℃。 </li><li>收集每个样品至少八个神经管半部，以确保足够的产量为下一步骤。池小样本，如果几个电/夹层会话都需要积累的神经管的足够数量。储存足够的样本进行重复或一式三份每个条件。 </li></ol> 3.净化RNA和图书馆准备了Illumina的测序<ol><li>取下-80℃储存样品置于冰上15-30分钟。继续解冻在室温下，直到完成。 </li><li>涡流井，并在室温离开5分钟。旋涡再次离心以15,000×g离心1分钟以沉淀不溶的物质。 </li><li>将上清转移到新管中并根据该总RNA分离试剂盒手册说明书进行RNA提取。在洗脱建议的最低量，以增加浓度。 注:后清理DNase处理建议，以避免在RNA测序数据集的潜在基因组DNA序列的污染。 </li><li>轻弹管混合内容并删除5微升的质量控制分析。剩余的样品储存于-80℃直至文库制备;这些样品应解冻一次，以避免RNA降解。 </li><li>运行在生物分析仪器纯化的RNA用RNA 6000纳米试剂盒; RIN值必须至少为7以上进行。 </li><li>生成使用Illumina公司兼容的RNA测序文库制备试剂库和复用多达4个图书馆的HiSeq仪器设置为产生100 bp的单一读取一条车道。如果可能的话，包括在相同的运行，以尽量减少潜在的技术工件的所有样本。 注:本应产生高达50万转每个样品，这是绰绰有余的，以获得范围为大多数基因13 EADS。 </li></ol> 4.银河公共服务器10比较RNA测序数据集。 注:服务器的存储空间是有限的，并比较多个样品实验，可能需要删除在滤波步骤中生成的中间文件。 <ol><li>在银河公共服务器（创建用户帐户<a href="https://usegalaxy.org/">https://usegalaxy.org</a> ）和FTP上传Illumina的数据集FASTQ格式。 </li><li>准备与FastqGroomer 14岁以下的NGS工具上传的文件:质量控制和操作菜单;如果质量得分已经编码PHRED + 33（桑格），跳过此步骤只需更改文件类型下的属性为每个数据集fastqsanger。 </li><li>通过分析在NGS的FastQC工具，每个数据集的总体质量:质量控制和操纵我NU。 注:应特别注意给予对每个碱基序列的质量和每个碱基序列的内容所得到的图形，因为这些将提供估计用于修剪的读出端的参数。 </li><li>修剪读取放弃3"使用的工具FASTQ质量微调 14岁以下的NGS低质量的数据结束:质量控制和操作菜单。设置该工具只处理3&#39;端的质量分数低于20。 </li><li>卸下接头序列读取从使用裁剪工具 （ <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ）在NGS:质量控制和操作菜单。设置为丢弃具有小于45 bp的左，输入所使用的适配器的顺序读取，并选择输出到包括剪切和非截取序列的工具。此外，选择不丢弃具有未知碱基序列，因为大多数的低质量的碱中的最后一个步骤中已经除去。 </l我&gt; <li>使用工具修剪序列 （ <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ）在NGS:质量控制和操作菜单，修剪第一基地的读取，以消除FastQC检测5&#39;序列偏置（通常为10）。 注意:在这一点上，它是重新运行FastQC并与前面的分析进行比较，以确定该数据集的质量得到了改善一个好主意。 </li><li>下载并解压缩最新的鸡基因组组装从Illumina公司iGenomes在UCSC基因组浏览器（ <a href="http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn">http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn</a> ），并上传了全基因组FASTA格式的文件和基因注释GTF格式文件使用下获取数据菜单中的工具上传文件银河系的历史。 </li><li>使用下对准TopHat2 15地图读出各样品的鸡基因组农工商超市:RNA分析菜单。选择使用从历史基因组（选择步骤4.7上传的FASTA文件）。 </li><li>建立预测转录使用袖扣 16岁以下的NGS每个样本:RNA分析菜单。选择要使用的参考注解为指导（选择步骤4.7上传的GTF文件），打开选项执行偏差校正 （从历史，从步骤4.7 FASTA文件），并使用多读正确 。 </li><li>合并预测转录使用Cuffmerge 16岁以下的NGS所有样本:RNA分析菜单。选择要使用的参考注释（GTF文件从步骤4.7）和序列数据（从历史，从步骤4.7 FASTA文件）。 </li><li>通过比较TopHat2使用Cuffdiff 16岁以下的NGS每个样品产生BAM文件:RNA分析菜单。在现场笔录中选择了我rged转录由Cuffmerge生成并打开选项执行偏差校正 （从历史，从步骤4.7 FASTA文件），并使用多读正确 。 </li><li>排序差异表达测试使用的工具排序的筛选下表中列13（Q值）数值上升和排序菜单。 注:基因/成绩单显示实验条件之间的显著差异表达将呈现最低的Q值。 </li><li>与覆盖图形直观地检查结果，使用该工具创建的基因组覆盖率 17 下BEDTools菜单BedGraph先转换BAM文件bedGraph格式。禁用报告地区无覆盖，选择将拼接的条目不同的时间间隔，并通过了标准化测序深度被比较的所有数据集的一个因素缩放范围。 </li><li>使用所产生的bedGraph文件转换为权贵格式工具假发/ BedGraph到大人物 （ <a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ）在转换格式菜单。上传要人文件作为在UCSC基因组浏览器（自定义轨道<a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ）为galGal4组件和搜索感兴趣的基因进行可视化的覆盖在所述的数据集。 </li></ol>

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The authors have nothing to disclose.

在这里，我们为鸡脊髓电后分析效果的准则。虽然DNA载体进行电穿孔是更频繁地用于过表达感兴趣的基因，还可以使用构建体编码显性阴性，quimeric蛋白或前体的siRNA，以产生基因功能敲低的条件19-21。事实上，从两个GAIN-和丧失功能的分析得到的转录概况比较可以指出差异表达的基因在相反的方式中的每个条件，因此具有潜在地发现更多的具体效果。 如任何方法下游?...

在活的生物体的基因研究经常使用的鸡胚为模型，因为在OVO电代表了一个快速和廉价的方式来部分地转染胚胎结构在体内与DNA构建1-5。编码含有荧光报告与感兴趣的基因，如pCIG 6或机动设备7在一起的一个双顺反子转录物表达载体，允许在所希望的区域转质量的快速验证处理的胚胎用于下游分析之前，在立体显微镜下。 在DNA测序的最新进展，现在有可能从使用RNA测序-8,9- RNA样本获得数字全转录组表达图谱。因此，代替费时方法，使只有几个基因产物平行，分析如原位杂交，免疫组织化学和qPCR，用于制备cDNA文库的高通量测序可以提供全转录组的信息。对每一个基因的表达水平的实验效果的观察又可以提供关于所使用的构建体改变了通路有力的见解。这是特别容易的，如果一个基因组装配所使用的生物体是可用的，从而鸡胚又是一个合适的模型。 如果用户有访问权限的可靠的基因组测序中心，主要问题是获得高品质的RNA样品。这项工作表明获得的RNA样品从转染的神经管适用于RNA测序，以及在下游步骤中所得到的数据集的计算机分析的方法。电在HH12-13其次是48小时培养后，转染树干脊髓半解剖的核糖核酸酶自由的条件下，立即溶解并进一步降解保护的超低冰点，直到RNA纯化和图书馆preparat离子。 银河公共服务器10可以访问免费资源，用于分析的高通量测序数据。的空间供注册用户的数量是足够的小实验，从而省去了本地的计算基础设施。 RNA测序数据分析包括过滤，比对和基因表达的定量/对比。虽然有用于RNA测序数据分析的一般准则，滤波参数将在很大程度上取决于序列的质量，并应后的原始数据的质量控制分析来确定。 这个协议描述的步骤，以获得与从鸡胚胎转染体内脊髓比较RNA片段轮廓。作为代表性的结果，从转染了空载体两个独立的对照样品获得的数据集的比较表明，该方法是可重复的，应该允许鉴定差异EXPRES的SED成绩单实验样本。因此，该方法的应用有可能大大增加的发现的数量之后，一个单独的试验，从而提供一个大的数据集进行后续调查。

<table><tbody><tr><td>Indian ink</td><td>Any supplier</td><td></td><td></td></tr><tr><td>18G x 1 1/2 needle</td><td>BD</td><td>305196</td><td></td></tr><tr><td>3 ml syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Tris</td><td>MP Biomedicals</td><td>819620</td><td></td></tr><tr><td>F D &amp;amp; C Blue 1</td><td>Spectra Colors Corporation</td><td>5.FC.0010P0</td><td>Food Dye used to visualize DNA injection</td></tr><tr><td>Ringer&#039;s solution (1 L)</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 7.2 g NaCl</td><td>Sigma-Aldrich</td><td>S-9888</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.37 g KCl</td><td>Sigma-Aldrich</td><td>P-4504</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.225 g CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>10043-52-4</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.217 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S-9390</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.02 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P-5379</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 800 ml and adjust pH to 7.4</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 1 L</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Filter and autoclave</td><td></td><td></td><td></td></tr><tr><td>PBS (Phosphate Buffered Solution) (1 L)</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 8 g NaCl</td><td>Sigma-Aldrich</td><td>S-9888</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.2 g KCl</td><td>Sigma-Aldrich</td><td>P-4504</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 1.15 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S-9390</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.2 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P-5379</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 800 ml and adjust pH to 7.2</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 1 L and filter</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 1 ml DEPC (Diethylpyrocarbonate)</td><td>Sigma-Aldrich</td><td>D-5758</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Shake vigorously and let stand overnight at room temperature</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Autoclave</td><td></td><td></td><td></td></tr><tr><td>Sieved spoon</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Sterile 60 x 15 mm polystirene Petri dishes</td><td>Corning Life Sciences</td><td>351007</td><td></td></tr><tr><td>RNaseZap RNase decontamination solution</td><td>Life Technologies</td><td>AM9780</td><td></td></tr><tr><td>Fine point surgical scissors</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Straight fine point tweezers</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Pulled glass needle made from 1.1 x 75 mm glass capillary tubes</td><td>Kimble Chase</td><td>40A502</td><td></td></tr><tr><td>9&#039;&#039; glass Pasteur pipette</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Manual pipette pump</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Clear 1.5 ml microcentrifuge polypropilene tubes</td><td>Corning Life Sciences</td><td>MCT-150-C</td><td></td></tr><tr><td>Microcentrifuge</td><td>Any supplier</td><td></td><td></td></tr><tr><td>RNAlater solution for RNA stabilization</td><td>Life Technologies</td><td>AM7020</td><td></td></tr><tr><td>RNAqueous total RNA isolation kit</td><td>Life Technologies</td><td>AM1912</td><td></td></tr><tr><td>Molecular Biology Grade Ethanol</td><td>Any supplier</td><td></td><td></td></tr><tr><td>RNA 6000 Nano Kit</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>2100 Electrophoresis Bioanalyzer Instrument</td><td>Agilent Technologies</td><td>G2939AA</td><td></td></tr><tr><td>Truseq RNA Sample Preparation Kit v2</td><td>Illumina</td><td>RS-122-2001/RS-122-2002</td><td></td></tr></tbody></table>

rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

为了验证所描述的方法，从胚胎电穿孔用空载体机动设备7和从胚胎用含有插入物构造SCRT2-ZNF，其编码鸡SCRT2 18的锌指结构域的相同向量中的一个样本中生成两个控制样品。样品得到11-22微克总RNA，每个从8至12中的胚胎池获得。所有三个样品中的生物分析仪（ 图1）进行的质量检查得分的10最佳的RIN值，这表明高品质的RNA可以与此协议来获得。 测序?...

Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

rna-seq-analysis-differential-gene-expression-electroporated-chick

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.5K 次观看.  Universidade de São Paulo. This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

视频: RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

科研

遗传学

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

发育生物学

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

通过CAS9蛋白-gRNA复合物电穿孔直接基因敲除Axolotl脊髓神经干细胞

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

神经科学

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

生物学

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

发育（青蛙）胚胎中的细胞谱系引导质谱蛋白质组学

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

化学

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...