我们感谢菲奥娜优良的实验室支持和斑马鱼的保健福利。这项工作是一个AHA博士前奖学金GW（11PRE5730027）的和NHLBI补助金的HJY（R01HL66292）和JDA（R01HL095690）。

概述特定阶段的斑马鱼胚胎注射液反义吗啉代寡核苷酸（MO），结合到靶mRNA和蛋白表达，转录干扰，被广泛用于基因敲除（损失函数）的研究斑马鱼13,14。基因的工具，LLC提供的MO，无论是羧基发出绿色荧光的或丽丝胺（发出红色荧光）的检测MO注射的胚胎中，使用荧光显微镜的标签。通过注入MO到蛋黄细胞在斑马鱼发育的不同阶段中，它能够提供的MO胚胎的具体的室中（ 图1A-F）。 MO注射成之间的蛋黄1-4细胞阶段（0-1 HPF）的进入所有胚胎细胞（ 图1A，D，D&#39;）， 通过连接与蛋黄的持续存在，直到32 -细胞阶段15，以促进全球击倒。 MO注射到蛋黄期间midblast乌拉阶段（2.5-3 HPF）的可以输入DFC的（ 图1B中，E，E&#39;）可能是通过胞质桥8和敲除基因的功能专门在DFC / KV细胞谱系12，而不进入大多数其它胚胎细胞谱系的祖。作为一个重要的控制测试基因功能是否需要在DFC / KV或也蛋黄12,16，但也可以限制密苏里蛋黄细胞注射后之间的圆顶30％外包阶段（〜4.5 HPF）的已经关闭了所有的胞质桥（ 图1C，F，F&#39;）。这些注射已被组合使用，以分析基因功能DFC / KV细胞17-24。为了评估中流体流量的KV，荧光微珠注入对KV管腔之间的6-10体节阶段（12-14高倍视野），然后立即使用电视显微镜（ 图1G-J）成像。微珠可发出红色或绿色荧光（或两者）的，所以它是可以使用不同的图像荧光微球和MO的渠道。 1。特定阶段的注射液的吗啉代（密苏里州） MO注射到斑马鱼的胚胎已被证明以前25,26。在这里，我们简要地描述特定阶段的MO注射。所有注射之后，胚胎转移到陪替氏培养皿，并在28.5℃的孵育<ol><li>全球MO注射:收集胚胎受精后，立即将其装入一个喷射板。荧光MO加载到毛细管针，针安装到微量注射器（ 如哈佛设备PLI-90的Pico-喷油器）。额的针尖和调整喷射设置（压力和/或时间），以创建具有一定体积的1 NL所描述25,26注射降使用解剖体视显微镜，注入1 NL到蛋黄的MO（ 图1A）的≥50胚胎每个实验。快速完成注射液的4芯雄鹿E。 </li><li> DFC的目标MO注射:收集后立即受精的胚胎和孵化为28.5°C。 ，当胚胎已经达到256个细胞的阶段（约2.5 HPF），快速加载的喷射板，然后注入到蛋黄≥100之间的256细胞和1000细胞阶段的胚胎（ 图 1 NL荧光MO 1B）。 </li><li>卵黄的目标MO注射:收集胚胎受精和孵化后立即为28.5°C。在圆顶阶段（〜4 HPF）的，加载的胚胎成喷射板，然后注入荧光MO 1 NL≥100胚胎之间的圆顶和30％外包阶段（ 图1C）的蛋黄。 </li></ol> 2。选择注射的胚胎分析<ol><li>当MO注射胚胎达到75％外包阶段（8 HPF），去除未受精和死胚，然后屏幕活胚胎在荧光显微镜分布的荧光MO。然后分配瓦特选择胚胎发育为28.5°C。 <ol><li>对于全球密苏里注射，选择胚胎荧光均匀地分布在整个所有的胚胎干细胞（ 图1D，图2A）。 </li><li> DFC目标MO注射，胚胎荧光MO已扩散到蛋黄，似乎集中在背胚率（DFCS）（ 图1E;图2B）。在这个阶段中，也可以是难以可视化荧光DFC的由于在底层卵黄明亮的荧光。小心排除胚胎的荧光MO纳入胚胎干细胞以外的其他八间指定流感诊所求诊或聚集在注射部位（ 图2D）。 </li><li>对于蛋黄目标MO注射，选择其中MO荧光均匀地分布在整个蛋黄并没有观察到任何胚胎干细胞（ 图1F，图2C）中的胚胎。同样，取出胚胎的荧光MO仍然聚集在注射部位。 </li></ol></li><li>之间的2-4-体节阶段（〜11 HPF）的，屏幕胚胎在荧光显微镜下，使用更高的放大倍率的第二时间。然后让选定的胚胎发育为28.5°C。 <ol><li>对于全球MO注射，确保选定的胚胎具有MO荧光均匀地分布在KV和所有的胚胎干细胞（ 图1D，图2E）。 </li><li>对于有针对性的DFC-MO注射，精心选择胚胎，MO荧光只有在KV细胞和蛋黄（ 图1E）。转基因胚胎表达GFP KV细胞，例如作为Tg（DUSP6:d2EGFP）27，可以帮助识别在其中MO已成功交付至KV细胞（ 图2F）的胚胎。放弃MO荧光另一方面，KV细胞在胚胎干细胞的胚胎。 </li><li>蛋黄目标MO注射，选择MO荧光的胚胎，只在蛋黄（ 图1F:图2G）。 </li></ol></li></ol> 3。安装胚胎在KV流体流动分析<ol><li>分析非对称流体流动的KV全球MO，DFC有针对性的MO或蛋黄目标MO注射胚胎，仔细dechorionate的〜20胚体节间4-6级（11-12 HPF）用锋利的镊子。 </li><li>准备到5ml管作为干浴中的液体保持在42℃下的50毫升的1％低熔点琼脂糖和等分试样</li><li>一个胚胎转移到玻璃抑郁症幻灯片（美国科学用品公司），并尽可能删除尽可能多的水。固定化的胚胎通过添加足够温暖的1％低熔点琼脂糖刚好盖住胚胎（太多琼脂糖可以干扰随后的成像）。琼脂糖凝固，使用产钳的位置，胚胎等，KV朝上（背视图）（ 图1H）。安装10每张幻灯片的一个胚胎的胚胎。快速地工作，以确保在下一步骤中的所有注射​​并发症eted由10个体节阶段（14 HPF）的。 </li></ol> 4。荧光微球注射到KV <ol><li>稀释Fluoresbrite Multifuorescent 0.5微米直径的微球（Polysciences的公司）在无菌水中至1:50在1.5ml Eppendorf管中。 </li><li> 1:50稀释的微珠混合彻底攻管和加载3微升毛细管针。使用的微量注射器用于MO注射（ 如哈佛设备PLI-90的Pico-注射器），打破了针尖，镊子和调整注入的设置，以产生尽可能小的（&lt;0.5 NL）注射下降。 </li><li>在解剖显微镜下，对齐与KV针内腔中的第一安装胚胎。将针头插入管腔和注入体积小（&lt;0.5 nl）的珠（ 图1G）。一个小针尖小的注射量是关键，以避免损坏KV。 </li><li>注射所有安装胚胎。一旦胚胎injecte的d，a将一滴无菌水可以添加琼脂糖上，以防止其干燥。珠粒的注射过程中，往往会阻碍针开口。这需要重新打破的针尖和调整通过调节压力或时间设定的显微注射装置的注射量。更换针头变钝，在注射过程中造成重大损害。 </li><li>屏幕注射的胚胎为成功交付珠一个堂堂正正的荧光化合物显微镜下（ 例如蔡司AxioImager M1）使用20倍的目标。首先，确定是否使用明视场照明（ 图1J）KV结构是完整的，然后用荧光观察珠粒。选择在珠是和里面的KV流明的胚胎。丢弃在KV KV损坏或不漂珠的胚胎。这是很容易会错过的KV，并提供附近的组织或相关蛋黄珠。如果不成功，安装ANOTHER 10个胚胎，并重复注射过程。 </li></ol> 5。 KV流体流量的可视化和分析<ol><li>对于每个选定的胚胎KV珠内，加一滴无菌水覆盖琼脂糖，然后使用63X水浸泡目标（ 图1I）直立荧光化合物显微镜下观察KV。可替换地，可以应用于用于与非水浸渍的目标和/或倒置显微镜盖玻片。 </li><li>使用高速摄像机安装在显微镜上（ 例如蔡司AxioCamHSm）录制10秒的电影。记录使用明场或微分干涉相差（DIC）的信道使用的荧光通道（约70帧/秒），和对KV管腔的珠粒。 </li><li>为了形象化所有珠的动作，随着时间的推移，进口电影的荧光珠ImageJ软件（免费下载<a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a> ）一的第二创建一个最大投影在电影中所有的荧光信号。要执行ImageJ的最大投影中，单击"图片→栈→Z项目。"在"Z项目"窗口中，项目全部与投影片类型的"最大强度"。此图像可以叠加在DIC的图像，其中珠粒成像（ 图3A-B）的KV。 </li><li>个人珠的变动可以使用ImageJ进行跟踪。可以下载一个插件（"手动跟踪"）在<a href="http://rsbweb.nih.gov/ij/plugins/track/track.html" target="_blank">http://rsbweb.nih.gov/ij/plugins/track/track.html</a> 。打开荧光微球在ImageJ的电影和运行的手动追踪功能。手动跟踪，在该窗口中，选中"显示参数"和"时间间隔"和"X / Y校正"的输入参数。手动选择保持≥50帧的电影在焦平面的珠子，然后跟踪≥5珠，每胎。每个胎圈的路径和速度所产生的次e软件（ 图3 CD）。 </li></ol>

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(25), e1115 (2009).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113 (2009).</li><li>Molina, G., 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=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat. Chem. Biol. 5 (9), 680-687 (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1995).</li><li>Schneider, I., Houston, D. W., Rebagliati, M. R., Slusarski, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+fluxes+in+dorsal+forerunner+cells+antagonize+beta-catenin+and+alter+left-right+patterning.">Calcium fluxes in dorsal forerunner cells antagonize beta-catenin and alter left-right patterning.</a> Development. 135 (1), 75-84 (2008).</li><li>Clement, A., Solnica-Krezel, L., Gould, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cdc14B+phosphatase+contributes+to+ciliogenesis+in+zebrafish.">The Cdc14B phosphatase contributes to ciliogenesis in zebrafish.</a> Development. 138 (2), 291-302 (2011).</li><li>Matsui, T., Bessho, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left-right+asymmetry+in+zebrafish.">Left-right asymmetry in zebrafish.</a> Cell Mol. Life Sci. , (2012).</li></ol>

没有利益冲突的声明。

使用特定阶段的目标MO注射到DFC / KV细胞系是一个非常有用的方法来研究基因功能的细胞自主性，避免多效性所造成的全球基因敲除的表型。然而，这些注射可以在技术上具有挑战性。注射液的MO之间的256细胞和1000细胞的阶段，可能会导致在三种可能的结果:1）在MO仍然聚集在注射部位，2）的MO整个蛋黄扩散并进入DFC / KV细胞或3）的MO整个蛋黄，进入DFC / KV细胞和胚胎干细胞的扩散。因此，在这个实?...

<table border="1"><tbody><tr><td>的试剂/材料名称</td><td>公司</td><td>目录编号</td></tr><tr><td>标准控制寡里沙明标记</td><td>基因工具，LLC </td><td></td></tr><tr><td>定制Rock2b吗啉代寡核苷酸</td><td>基因工具，LLC </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent 0.5微米的微球</td><td> Polysciences的公司</td><td> 24054 </td></tr></tbody></table>

analysis of gene function and visualization of cilia-generated fluid flow in kupffer's vesicle

内部器官，如心脏，大脑和肠道左右（LR）为他们的正常功能是至关重要的不对称性。运动纤毛参与建立LR的不对称性在脊椎动物胚胎，包括老鼠，青蛙和斑马鱼2-6。这些&#39;LR的纤毛&#39;产生不对称的流体流动是必要的，以触发一个保守的不对称交点（TGF-β超家族）的信号级联在左侧侧板中胚层，这被认为是提供开发机关7 LR构图信息。因此，理解机制LR构图，它是必不可少的，以确定基因调节的LR纤毛细胞的组织，运动和长度LR纤毛和他们的能力，以产生强大的非对称流。 在斑马鱼胚胎中，LR纤毛位于巨噬泡（KV）2,4,5。 KV是由一个单一的层monociliated上皮细胞，附上一个充满液体的腔。 KV是来自一组被称为的背先行者细胞（DFCS）迁移外包阶段在背胚率在8,9〜20-30细胞的命运映射。在早期的体节阶段，DFC的集群，并分化成纤毛上皮细胞，形成KV中的胚胎10,11 tailbud。结合的光学透明度和快速发展的斑马鱼胚胎斑马鱼KV一个很好的模型系统来研究，LR纤毛细胞能够识别和跟踪八间指定流感诊所求诊。 有趣的是，DFC / KV细胞谱系的祖细胞保留蛋黄细胞的细胞质之间的桥梁，受精后4小时（HPF）的，而蛋黄细胞和其他接近8高倍视野2后的胚胎干细胞的细胞质之间的桥梁。利用这些细胞的桥梁，我们开发了一个特定阶段的喷油策略提供吗啉代寡otides（密苏里州）专门DFC的和击倒的目标基因的功能，在这些细胞中12。这技术创造嵌合体胚胎基因的功能被撞倒在发展的背景下，野生型胚胎的DFC / KV血统的。在KV，我们要分析非对称流体流动注射荧光微球到KV珠流明，并记录运动时，通过电视显微镜2。流体流动是易于可视化和可量化的跟踪珠位移随着时间的推移。 在这里，使用特定的阶段，的DFC-针对性的基因敲除技术和注射荧光微球KV可视化流程，我们提出了一个协议，提供了一个有效的方法来描述一个特定基因的作用在KV的发育和功能。

图1给出了一个流程图，用于测试基因功能DFC / KV细胞和如何引入荧光珠的可视化流体流的喷射策略阶段特异性的MO注射提供了一种有用的方法来分析基因功能的特定的室中的胚胎。在KV。在成功阶段特异性注射胚胎的是，荧光的MO分布示意性地示出在图2中，在图1D-F和在活胚胎。一个不成功的MO注射，在其中的MO仍然在蛋黄细胞聚集， 如图2D</...

Watch this Scientific Journal Video about Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle at JoVE.com

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

纤毛产生的流体流动在枯否囊泡（KV）控制的斑马鱼胚胎的左右模式。在这里，我们描述了一种技术，调节基因的功能，特别是在KV细胞。此外，我们展示了如何提供KV可视化流体流动的荧光珠。

基因功能分析和可视化的纤毛产生的流体流动的在枯否囊泡中

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

analysis-gene-function-visualization-cilia-generated-fluid-flow

Research

JoVE Journal

Biology

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

14.8K 次观看.  Upstate Medical University. 纤毛产生的流体流动在枯否囊泡（KV）控制的斑马鱼胚胎的左右模式。在这里，我们描述了一种技术，调节基因的功能，特别是在KV细胞。此外，我们展示了如何提供KV可视化流体流动的荧光珠。

视频: Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

科研

生物学

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和"剪切环"中的应用:一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

西普库鲁斯核素体液的原位杂交

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture method to obtain a sufficient number of LG stem cells (LGSCs) is one obstacle for further research and application. The three-dimensional (3D), serum-free culture method for adult mouse LGSCs is well established and shown here. The LGSCs could be continuously passaged and induced to differentiate to acinar or ductal-like cells.
For the LGSC primary culture, the LGs from 6-8-week-old mice were digested with dispase, collagenase I, and trypsin-EDTA. A total of 1 &#215; 104 single cells were seeded into 80 &#181;L of matrix gel-lacrimal gland stem cell medium (LGSCM) matrix in each well of a 24-well plate, precoated with 20 &#181;L of matrix gel-LGSCM matrix. The mix was solidified after incubation for 20 min at 37 &#176;C, and 600 &#181;L of LGSCM added.
For LGSC maintenance, LGSCs cultured for 7 days were disaggregated into single cells by dispase and trypsin-EDTA. The single cells were implanted and cultured according to the method used in the LGSC primary culture. LGSCs could be passaged over 40 times and continuously express stem/progenitor cell markers Krt14, Krt5, P63, and nestin. LGSCs cultured in LGSCM have self-renewal capacity and can differentiate into acinar or ductal-like cells in vitro and in vivo.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

用于泪腺干细胞的三维无血清培养系统

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

通过纳流式细胞术表征单个细胞外囊泡跨膜蛋白

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...