Research supported by Alberta Livestock and Meat Agency, Alberta Innovates - BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.

这项研究的动物部分在阿尔伯塔省埃德蒙顿，加拿大大学的代谢研究单位进行，由亚伯达省动物护理和使用委员会大学所有的动物实验程序批准（协议＃AUP00000131），和动物的照顾根据以动物保健指南加拿大委员会（1993年）。 1.胚胎生产，总RNA和DNA酶处理的分离<ol><li>对于动物实验方案和胚胎收集参阅在我们以前的出版物17 和 18。 </li><li>池和捕捉来自在液氮每头奶牛冷冻类似阶段的胚胎，然后在保持-80℃直至RNA提取。 </li><li> 通过基于列的RNA提取试剂盒使从微微规模的样本提取RNA（包信息，请在物料清单）中提取总RNA。 注:推荐套件包括:调节缓冲液，提取BUFFER，70％乙醇，洗涤缓冲液1中，洗涤缓冲液2中，洗脱缓冲液，以收集管和离心管RNA纯化列。 </li><li>添加10μL的提取缓冲液含有胚胎管中并在42℃孵育30分钟。在800 XG 2分钟的离心管收集细胞提取物到离心管中。 </li><li>吸取250μL调理缓冲到纯化柱过滤膜。孵育调理缓冲RNA的纯化塔，在室温下5分钟。离心在收集管中的纯化塔以16,000 xg离心1分钟。 </li><li>吸移管10微升70％乙醇的成RNA提取缓冲液和胚胎的混合物。吹打上下拌匀。吸取混合气进入预处理纯化柱。 </li><li>以100 xg离心结合的RNA，离心纯化塔2分钟，通过离心16,000 xg离心紧接30秒通过除去流动。 </li><li>吸取10081，L洗涤缓冲液1到纯化塔和离心机在8000×g下1分钟。 注意:DNA酶处理建议后，是否提取RNA进行逆转录或扩增。 </li><li>步骤1.8之后消化基因组DNA。 注:建议DNA酶试剂盒在材料列表中。推荐套件包括:DNA酶I原液和缓冲器。 </li><li>通过混合5微升的DNA酶我原液35μL缓冲准备的DNase孵育混合物中。轻轻倒转混合。 </li><li>直接吸取40微升的DNA酶保温混合物进入纯化柱膜。在室温下孵育15分钟。 </li><li>吸取40μL洗涤液1到纯化柱膜上，然后离心在8000 XG为15秒。 </li><li>吸取100μL的洗涤缓冲液2进入净化塔和离心机在8000×g下1分钟。 </li><li>吸取另外100微升洗涤缓冲液2到纯化塔和离心机为16,000×g的2分钟。 </li><li>纯化塔转移至新的0.5毫升离心管中。吸移管11洗脱缓冲液的微升直接在纯化塔的膜。以确保洗脱缓冲液的最大吸收到膜中，吸移管的尖端轻轻触碰到膜的表面，而分配所述洗脱缓冲液。 </li><li>孵育柱在室温下1分钟。离心1分钟列在1000 xg离心到列分发洗脱缓冲液，然后在16,000g XG旋转1分钟以洗脱RNA。 </li><li>根据制造商的说明来评估RNA的质量和数量19使用生物分析仪。 </li><li>储存在-80℃直到使用RNA样品。 </li></ol> 2. RNA扩增和标记的微阵列分析注:对于RNA扩增和标记程序工作的第一次使用，使用五个NG穗在RNA ALO与实际的aRNA样品ng到监视RNA扩增和杂交的质量控制测量。在穗的RNA结构包含10个在体外合成，在预定比例多聚腺苷酸的成绩单。当程序适当地进行，标记的转录物特异性只杂交到阵列互补控制探针和数据可以被扫描以跟踪具有最小自或交叉杂交的质量控制的保证。关于扫描阵列和数据分析后的尖刺，在控制强度和规范化步骤的详细介绍请参考以前的出版物20，21。下面的过程是考虑到常规微阵列的用户。 <ol><li> 11微升 - 在10总体积制备100pg的高品质的RNA样品（RNA完整性数（RIN）值至少&gt; 7.0）。 </li><li>扩增RNA样品与扩增试剂盒能够有微微的样本规模的工作（低至100 PG）。 注:推荐扩增试剂盒信息，请在材料清单，并遵循制造商的说明书没有任何修饰。 </li><li>使用基于荧光的定量评估扩增的aRNA的大小范围的分布。 </li><li>使用分光光度计仪器（基于在260和280nm的吸光度）来测量扩增的aRNA的浓度。 </li><li>存储放大阿尔纳在-80°C，直到准备标签。 </li><li>根据ULS阿尔纳标记试剂盒用户指南使用放大RNA 2微克的荧光标记。 注意:由于花青547和花青647染料是光降解和花青647染料敏感容易被臭氧分解，标记过程中发生的光的最小量和无臭氧环境下。 </li><li>打开臭氧箱（ 图1A），直至臭氧的电平为0.001 ppm的。 </li><lI&gt;通过加入花青547或花青647，标记缓冲液2微升2微升执行臭氧箱（ 图1B）内的反应设置，然后调整至20μL的无RNA酶的水的安全灯下的最终体积。暗室过滤器添加到所述光源。 </li><li>轻轻混匀，并在85°C孵育在热循环仪的管15分钟。在冰上至少1分钟发生的样本。降速用RNA提取试剂盒出发，除了相关的DNA酶处理（1.9至1.11）的步骤之前，收集管的内容，以清理CY-染料标记的扩增的RNA。 </li><li>使用分光光度计仪器来测量和确定标记的扩增的RNA的浓度并在使用前保持标记的aRNA在-80℃下最大为3天。 </li></ol> 3.基因芯片杂交，洗涤注:以下重要步骤是根据双色微阵列基因表达分析协议改编（版本6.5，2010年5月）。 <ol><li>添加从每个菁547-和647菁标记的aRNA纳克的825等量和25X碎片和10倍的封闭液混合。 </li><li>加热该混合物在60℃下15分钟，并在冰上冷却1分钟。 </li><li>加入2X杂交缓冲液等量拌匀。 </li><li>离心机以13000rpm，然后将混合物已准备好用于杂交。 </li><li>从该混合物中加载100微升到阵列滑动并密封杂交室内部的幻灯片。 </li><li>在65℃下装入装配室分成杂交烘箱17小时在烤箱10的转速旋转。 </li><li>根据浸入稳定和30秒干燥的解决方案与臭氧保护治疗方案洗净阵列。 </li><li>从溶液中取出阵列并确保该阵列表面干燥，无粉尘颗粒</li></ol> 4.扫描微阵列<ol><li>保持阵列在黑暗的地方，打开scann呃，直到它准备使用（15分钟等待时间）。 </li><li>加载阵列条形码左，进入扫描仪和开始扫描。根据软件用户指南执行现场分析并保存数据（GPR）文件格式。 </li></ol> 5.统计分析<ol><li>下载软件FlexArray http://genomequebec.mcgill.ca/FlexArray/license.php点击"导入原始数据"和GPR文件加载到FlexArray。 </li><li>如有必要，通过之后的指令执行正规化和统计分析。注:因为以前22说明计算在这项研究中积极选点的阈值。 </li><li>选择下拉从FlexArray的情节观众手动查看所有杂交斑点和背景信号。注:杂交到芯片飙升后在控制的类似的图像可以在以前的研究中8可以看出。 </li><li>选择一个从FlexArray阵列正常化进程之间的分位数以下阵列内黄土正常化相应下拉手册。 </li><li>从导出所有FlexArray归一化数据到一个Excel文件以便今后使用。 </li></ol> 6.生物信息学分析从牛胚胎特异性转录的探针序列的原始注解（BESTv1）微阵列先前已8所述。在本研究中，获得了20403独特的基因符号（GS）（ <a href="http://ecsource.jove.com/files/ftp_upload/53754/Supplement_file_1.xlsx">补充文件1</a> ）通过EmbryoGENE实验室信息管理系统（LIMS）ELMA（http://elma.embryo-gene.ca）。的6765独特的基因（名单<a href="http://ecsource.jove.com/files/ftp_upload/53754/Supplement_file_2.xlsx">补充文件2</a>被选中），对应于芯片正常化进程后，所有的积极信号（第5步.5）从牛7天胚胎基因表达谱。积极的信号阈值是基于从A值的信号强度透水出版16计算。 <ol><li>执行与PANTHER功能分析（蛋白质分析，通过进化关系）分类系统23，24，打开下面的链接（http://www.pantherdb.org/about.jsp）。 </li><li>上传的6765胚胎特异性GS列表使用统计人数过多试验胚胎发育过程中识别特定的生物过程，并使用普通牛的默认设置（在数据库中的所有基因）作为参考名单25。 注意:这允许从在统计上过高或过低表达用二项式检验的GO术语发育有关的过程的鉴定。 </li><li>在&lt;0.05设置P值阈值的软件默认。数据可以下载并博览会rted到Excel文件中进一步选择16。 </li></ol>

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Dev. 76, 762-772 (2009).</li><li>Vallee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+bovine+embryo+transcript+profiles+during+early+in+vivo+embryonic+development.">Revealing the bovine embryo transcript profiles during early in vivo embryonic development.</a> Reproduction. 138 (1), 95-105 (2009).</li><li>Dafforn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+mRNA+amplification+from+as+little+as+5+ng+total+RNA+for+global+gene+expression+analysis.">Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis.</a> Biotechniques. 37 (5), 854-857 (2004).</li><li>Beaujean, N., Jammes, H., Jouneau, A., Dufort, I., Rovert, C., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Reprogramming.">Nuclear Reprogramming.</a> Studying Bovine Early Embryo Transcriptome by Microarray. , (2015).</li><li>Patterson, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+comparison+of+one-color+and+two-color+platforms+within+the+MicroArray+Quality+Control+(MAQC)+project.">Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.</a> Nat. Biotechnol. 24 (9), 1140-1150 (2006).</li></ol>

The authors have nothing to disclose.

第一个问题使用7天的牛胚胎是没有得到足够数量的高质量RNA是研究基因表达进行微阵列分析。不推荐用于第7天的胚胎传统苯酚/氯仿RNA提取和乙醇沉淀法，导致低产量以及可能剩苯酚抑制RNA扩增反应。相反，一个标准的基于列的方法是更好地隔离总RNA，然后洗脱以最小的洗脱缓冲液，以增加浓度的RNA。在目前的研究是利用相同的方法学26，目前所使用的相同的RNA提取试剂盒<sup cla...

在高产奶牛胚胎早期损耗是在乳品行业1，2的主要挑战之一。牛已经成为一个有趣的模型来研究人类早期胚胎发育，由于其相似的发展过程3,4。然而，需要更多的研究来有关于涉及牛胚胎早期发育的基因更好的理解。 后由于第一微阵列技术二十年1995年5开发的，更为复杂的探测器制造工艺降低打印错误和内和不同微阵列平台6之间的阵列芯片可变性的发展。改进的微阵列技术导致了在临床研究7和广泛应用这种技术最近，在早期胚胎quality评估8。 大量的芯片技术所需的材料也就是为什么微阵列技术最初未能进入的一些研究领域，如早期胚胎发育的主要原因。最近，RNA扩增方法已得到改进，线性放大RNA为从起始原料RNA 9个子纳克微克的水平。目前市场上可用的几个商业RNA扩增试剂盒;然而，更多的受欢迎发达试剂盒相关的核糖单引物等温扩增10和T7启动子驱动的11种方法。最流行的反义RNA扩增的体外转录采用用寡dT引物在5&#39;端12连结到一个T7启动子。该技术允许保持了最具代表性的反义转录物的线性扩增后˚F或阵列杂交13。该方法已适于放大来自牛胚胎8提取的总RNA皮克水平。 万向联动系统（ULS）是直接结合的DNA或扩增的RNA与铂联荧光色素或者花青547或花青647，通过形成于鸟嘌呤14的N7位置的配位键的标记方法。该方法适于在胚胎的研究，以产生更稳定的相比改性酶法15产生的aRNA的氨基烯丙基扩增的aRNA无需修改。单染料和两种染料标记方法已经在芯片使用通用联动系统调整。一个大型芯片的比较研究是有数据质量的一个和两个彩色阵列平台6之间的良好的相关性。 近来，T7启动子驱动Ñ反义RNA扩增和ULS标记方法已被开发，以提供更可靠的协议来产生标记的aRNA材料用于微阵列杂交8,16的高品质的足够量。因此，这项研究提供了一个协议来演示一些从RNA提取使用7天牛胚胎作为一个例子涉及双色微阵列数据分析的重要步骤。

<table><tbody><tr><td>PicoPure RNA Isolation Kit</td><td>Applied Biosystems</td><td>KIT0204</td><td></td></tr><tr><td>RNase-Free DNase Set (50)</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Agilent RNA 6000 Pico Kit</td><td>Agilent Technologies</td><td>5067-1513</td><td></td></tr><tr><td>Arcturus RiboAmp HS PLUS Kit</td><td>Applied Biosystems</td><td>KIT0505</td><td></td></tr><tr><td>2100 Bioanalyzer Instruments</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>RNA Screen Tape</td><td>Agilent Technologies</td><td>5067-5576</td><td></td></tr><tr><td>ULS Fluorescent Labeling Kit</td><td>Kreatech Diagnostics</td><td>EA-021</td><td></td></tr><tr><td>Custom Gene Expression Microarrays</td><td>Agilent Technologies</td><td>G2514F</td><td></td></tr><tr><td>Agilent Gene Expression wash buffer 1</td><td>Agilent Technologies</td><td>Part #5188-5325</td><td></td></tr><tr><td>Agilent Gene Expression wash buffer 2</td><td>Agilent Technologies</td><td>Part #5188-5326</td><td></td></tr><tr><td>2x Hi-RPM Hybridization buffer</td><td>Agilent Technologies</td><td>&amp;nbsp;Part #5190-0403</td><td></td></tr><tr><td>25x Fragment buffer</td><td>Agilent Technologies</td><td>Part #5185-5974</td><td></td></tr><tr><td>10x GE Blocking Agent</td><td>Agilent Technologies</td><td>Part #5188-5281</td><td></td></tr><tr><td>Stabilization and drying solution</td><td>Agilent Technologies</td><td>&amp;nbsp;Part #5185-5979</td><td></td></tr><tr><td>Gasket slides enabled by Agilent SureHyb techonolgy</td><td>Agilent Technologies</td><td>G2524-60012</td><td>Pack of 20 gasket slides, 4 microarrays/slide</td></tr><tr><td>Two-Color RNA Spike-In Kit</td><td>Agilent Technologies</td><td>&amp;nbsp;Cat# 5188-5279</td><td></td></tr><tr><td>GenePix 4000B array scanner</td><td>Molecular Devices</td><td>GENEPIX 4000B-U</td><td></td></tr><tr><td>Ozone Free Box</td><td>BioTray</td><td>OFB_100x200</td><td></td></tr><tr><td>GAL file</td><td>Agilent Technologies</td><td>-</td><td></td></tr></tbody></table>

transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

从第7天的牛胚胎的总RNA和扩增的aRNA的代表性结果示于图3和表1中总结。 RNA的完整性和轮廓可以提取RNA后进行评估。 RNA的质量评估可以通过生物分析仪（ 图3A），并只与RIN值的那些样本高于7.0有资格被用于扩增（ 表1）来完成。 ?...

Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

transcriptome-profiling-vivo-produced-bovine-pre-implantation-embryos

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

7.7K 次观看.  University of Alberta. Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

视频: Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

科研

发育生物学

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

激光辅助畜禽受精卵细胞质显微注射

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

遗传学

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo&#39;s sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

30日猪胚胎的准确和无酚DNA性别鉴定的PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

功能性克隆使用<em&gt;爪</em&gt;卵母细胞表达系统

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

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 ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

转录组分析]

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荧光蛋白的表达