作者感谢彼得·鲁宾斯坦的有益的讨论，并提供技术咨询和大卫Pellman原PB1996克隆。这项工作是由March of Dimes的赠款，并从乘驾为孩子的资金支持。

1，克隆入质粒PB1996 <ol><li>从寡核苷酸制造公司，侧翼的靶序列，并包含在所选择的亲本质粒独特的限制性酶切位点的设计和秩序的DNA引物。注意:在这种情况下，设计引物以扩增400个碱基对的AIP1序列的5&#39;末端。该XhoI限制性位点掺入引物约20 bp的前AIP1序列，XmaI被包含在目标AIP1序列后约20个基点。用于克隆的质粒是PB1996，其中包含一个3XGFP标记为荧光，一个URA基因的酵母菌株的选择，和氨苄青霉素抗性基因对细菌的选择。 </li><li>净化从浦 - 酵母单倍体菌株的基因组DNA。注:请参考图1进行克隆过程的示意图。 </li><li>使用两个引物和酵母基因组DNA扩增Aip1p基因与在任恩的限制性酶切位点上运行一个标准的PCR反应ð。消化PCR产物和PB1996质粒在每个制造商独立的反应限制性内切酶推荐的协议。在这种情况下XmaI和XhoI，在37℃下进行1小时使用所附缓冲液中。 </li><li>跑0.8％琼脂糖凝胶电泳分离消化的DNA片段。在一个良好，加载一个低质量的DNA阶梯，在不同的孔，装入每个消化整个反应。运行该凝胶在100伏约1小时，直到染料前沿接近凝胶的末端。可视化的凝胶在UV光并纯化出对应于该蛋白片段的链段和切质粒。 </li><li>结扎消化AIP1段和PB1996质粒。变换成产品制造商使用的协议（10微升的DNA用100微升的细胞在冰上，热休克30分钟，在42℃进行2分钟，2小时，在37℃生长在SOC培养基）和平板上培养板DH5α细胞包括抗生素选择，在这种情况下，LB +功放板。选择并在液体培养基中生长一个菌落。纯化的新设计的质粒。 </li></ol> 2，突变酵母菌株产生<ol><li>使用，其中肌动蛋白基因已被删除构建突变酵母菌株单倍体酵母菌株。注意:在这种情况下，亲本菌株是从彼得·鲁宾斯坦博士（爱荷华大学）一份厚礼，建筑是由库克等人 20描述这株缺乏染色体肌动蛋白基因，并包含一个质粒编码的野生型酵母。肌动蛋白和尿嘧啶。 </li><li>使用PRS质粒编码的野生型酵母肌动蛋白与色氨酸+选择为基础的质粒诱变21。 </li><li>在PRS质粒进行定点突变工程师的错义突变成肌动蛋白。 <ol><li>为R256H突变的肌动蛋白，使引物的5&#39;ggtaacgaaagattccatgccccagaagc 3&#39;的Arg 256更改为他的256。运行一个标准的诱变与来自步骤2.2的质粒的PCR反应。 </li><li>从PCR改造的突变体肌动蛋白的质粒转化DH5α细胞。分离质粒DNA并测序的肌动蛋白基因，以验证该突变。 </li></ol></li><li>变换编码突变酵母肌动蛋白为来自步骤2.1的单倍体酵母菌株的质粒。 </li><li>文化对TRP-板的转化细胞来选择含有含有突变的酵母肌动蛋白的质粒色氨酸+酵母。 </li><li>选择细胞缺乏，通过从在包含5 - 氟乳清酸（5-FOA）的平板色氨酸板传代培养的细胞编码的野生型肌动蛋白和尿嘧啶（在2.1中所述）的原始质粒中。 5-FOA转化为有毒的形式，5 - 氟尿嘧啶，在yeaS的菌株表达功能URA3基因。逐步选择后生长的细胞只含有突变的肌动蛋白的质粒。 </li><li>纯化的新的酵母菌株的质粒DNA。序列的质粒，以确保该突变的肌动蛋白基因的存在。使用这个S训练中进一步研究。这个过程是在图2。 </li></ol> 3，转变成质粒酵母细胞<ol><li>消化Aip1p-GFP的质粒用限制性内切酶，以允许整合到酵母染色体中。注:限制性酶切位点必须是包含荧光标记，目的基因和选择标记的序列块外。在这种情况下，使用1微升高致病性禽流感限制性内切酶与8μLDNA和1微升的陪同10x缓冲液1小时，在37°C。 </li><li>文化学酵母细胞（来自步骤2.5），它们无法在车轮过夜合成尿嘧啶（URA-菌株）在5毫升的YPD培养基（1％酵母提取物，2％蛋白胨，2％葡萄糖），于30℃下降速将1ml细胞5分钟的1000×g的。 </li><li>制版混合物。使10XTE加入5毫升1M的Tris pH值7.5和2ml的250mM的乙二胺四乙酸（EDTA）以43毫升双蒸2 O的添加00.204克的乙酸锂至20ml 1XTE的，然后按8克聚乙二醇（PEG）（分子量〜3300）和过滤消毒。 </li><li>从在步骤3.2离心下来的细胞弃去上清，重悬的细胞中残留的液体。以悬浮细胞，加入2微升10毫克/毫升鲑鱼睾丸载体的DNA。加10微升来自步骤3.1和涡流消化的质粒DNA。加入0.5毫升板和旋涡。加入20微升1M的DTT和旋涡。 </li><li>孵育6-8小时，将混合物在25℃下热休克的细胞在水浴中于42℃10分钟。板100微升的微量离心管中，他们已经解决了上一浦板的底部的细胞。孵化在30℃2-3天，或直至菌落形态。 </li><li>选择单个菌落和条纹出到浦板块。注意:这些细胞含有与AIP1-GFP和浦基因整合到染色体中的质粒，允许增长。这些细胞然后在microsco使用PY。 </li></ol> 4，可视化Aip1p蛋白质运动使用全内反射荧光显微镜<ol><li>生长的酵母细胞与结合Aip1p-GFP在YPD培养过夜的车轮在30℃下继代培养将1ml过夜培养成9毫升浦介质。生长的细胞额外3-4小时在摇床上于30℃，以确保它们处于对数生长期。 </li><li>加入3微升细胞至玻璃显微镜载片上，并加盖玻片。使细胞定居在幻灯片上5分钟。将滑动舞台板的支架。 </li><li>观察Aip1p-GFP蛋白与全内反射荧光（TIRF）显微镜（488纳米），使用油浸渍100X TIRF物镜和一个数字CCD照相机。使用SlideBook 5.0软件，获取图像，持续20秒以5帧/秒的（获取的图像的各个帧被示于图2A）的速率。 <ol><li>把重点放在了C英语学习者，开放SlideBook 5.0软件，然后单击工具栏上的焦点的窗口按钮，进入重点监控。选择范围选项卡，然后选择100X-TIRF目标。打开灯，灯条滑动到15％。更改彬设置以2×2，改变过滤器明场。 </li><li>在显微镜下，使用精细调焦旋钮，使细胞成为关注的焦点看到电脑屏幕上。 </li><li>使用对焦窗口中的控件，可将灯泡熄灭，更换过滤器到Live，然后单击TIRFM按钮。 </li><li>在连接到显微镜的右侧端口的TIRFM照明器，开启激光发射为开。 </li><li>选择焦点的窗口内的流选项卡中，选中该框旁边开始录制，然后单击亮场按钮。 </li><li>在TIRFM照明器，转动千分尺，以调整激光的入射角度。这是用来从AIP1-GFP荧光点优化的荧光发射和从尽量减少背景荧光细胞和幻灯片。 </li><li>当显示所需的图像，然后按开始键。经过20秒，按停止。保存图像。在主菜单栏上的文件选项卡上，选择保存幻灯片作为并输入文件的位置和名称。 </li></ol></li><li>使用ImageJ的软件分析图像。使用宏插件"手动跟踪器"追踪荧光Aip1p灶的运动和速度。时间间隔参数更改为0.2秒。 <ol><li>使用add曲目选择，选择并通过七时五十六结缔组织帧跟踪单个荧光蛋白。使用结束track命令和每帧之间的运动蛋白质的速度将在结果窗口中显示。 </li><li>确定使用的是Microsoft Excel电子表格的每一帧之间的平均速率。使用该值来计算Aip1p运动的平均速度为每个感兴趣的细胞株。注:Aip1p运动和平均非线性速度率的散点图如图FIGU重新2B。 </li></ol></li></ol>

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以可视化的细胞骨架和实用程序的动态，对致病基因突变研究一种有效的策略已经在这里描述。先进的成像方式创造了新的机会，了解蛋白质在细胞内的运动靠近细胞膜。全内反射荧光显微镜（TIRF）是一种灵敏的技术用于在活细胞中的功能性研究。 TIRF使用创建由于在盖玻片和水性介质的折射率差的渐逝场的角度的激励激光器。渐逝场的能量激发荧光团，但与盖玻片和水之间的界面的距离呈指数?...

肌动蛋白是一种包括细胞骨架的主要蛋白质，参与关键的细胞过程，包括细胞分裂，细胞器运动，细胞运动，收缩和信令。在过去的十年中，致病突变的肌动蛋白被发现在每一个六人肌动蛋白异构体，导致一系列的疾病的，从肌病冠状动脉疾病1-7。由肌动蛋白基因突变导致疾病的进程继续阐明。酵母模型仍然是金标准，研究由于单一基本肌动蛋白异构体，遗传易处理性和高保护肌动蛋白的序列和功能的优势在肌动蛋白功能的突变的生化效应。研究表明，个体的肌动蛋白基因突变导致分子的具体功能障碍与显性负效应8。例如，在γ-非肌肉肌动蛋白耳聋致病突变影响赖氨酸-118残基通过改变调节肌动蛋白结合蛋白ARP2 / 3 9。研究经常采用体外分析蛋白质:蛋白质相互作用。调查肌动蛋白突变对细胞生物学和，特别是肌动蛋白结合蛋白定位于细胞的效果是有限的。 体内的酵母细胞骨架的研究传统上依赖于固定细胞的倒置荧光显微镜10的图像。这些实验提供了约肌动蛋白细胞骨架的形态学基础数据。调查已自掺入三维共焦成像可视化的复杂的细胞骨架网络11，12。该成像允许肌动蛋白斑块和长丝的数量和相对位置的量化。薄膜部电子断层扫描已用于图像相对于保藏的亚细胞结构13致密的丝状网络的形态。拥挤的蜂窝s步具有小的横截面中可以检查细节与此技术。成像研究已经扩展到使用时间的推移荧光显微镜活细胞。当光漂白和背景荧光可以缓和，时间的推移成像可以研究到细胞骨架蛋白的动态和应对环境条件11，14。另外， 在体外微丝的动态可视化垫付引进全内反射荧光（TIRF）显微镜。相比宽视场显微镜，TIRF具有减少背景荧光的优点和增强的对比度以监测单丝15，16。凭借这些优势，全内反射荧光显微镜已经适应了细胞生物学家监测细胞结构在质膜17，18。细胞事件，包括改变的细胞骨架，可以被可视化的实时低光毒性，最大对比度和最小的背景荧光19。 为了更好地理解对移动，本地化和营业额在细胞内，全内反射荧光显微镜和蛋白标记细胞骨架蛋白肌动蛋白基因突变的影响进行了使用。这里，方法来研究在肌动蛋白上的在酿酒酵母中的细胞骨架动力学临床相关突变的影响进行说明。具体地说，肌动蛋白结合蛋白，Aip1p的定位和移动，被可视化和量化，在细胞中表达的R256H突变的肌动蛋白。这些技术配合体外生化研究，并允许蛋白质相互作用和功能的更深入的了解。

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aip1p dynamics are altered by the r256h mutation in actin

在肌动蛋白基因突变引起一系列由于特定的分子变化，往往改变细胞骨架功能的人类疾病。在这项研究中，荧光的成像标签的使用总的内部荧光（TIRF）显微镜是用于可视化和量化改变的细胞骨架动力学的蛋白质。 TIRF显微镜和使用荧光标记的也允许在肌动蛋白的突变引起的变化，细胞骨架动力学的定量。使用这种技术，在活细胞中的细胞骨架功能的定量有价值的补充在蛋白质功能的体外研究。作为一个例子，影响肌动蛋白残基R256的错义突变已经确定在3人肌动蛋白异构体表明该氨基酸起着调节的相互作用中起重要作用。利用酵母模型的骨架运动的肌动蛋白基因突变R256H的影响进行了研究。蛋白质，AIP1，这是众所周知的协助丝切蛋白在肌动蛋白解聚，是具有标记的绿色荧光蛋白（GFP）在N-末端和使用TIRF显微镜追踪体内 。 Aip1p运动的野生型和突变株的速率进行定量。在细胞中表达R256H突变的肌动蛋白，Aip1p运动被限制，并且移动速度是近一半在野生型细胞中测得（0.88±0.30微米/秒，R256H细胞相比，1.60速度±0.42微米/秒在野生型细胞中，对&lt;0.005）。

一种方法，以图像中的细胞骨架蛋白的动态呈现。肌动蛋白结合蛋白，Aip1p，被标记为绿色荧光蛋白。设计用于编码标记产物的质粒示于图1中。然后将所述质粒转化到酵母细胞中。的荧光的表达标签Aip1p允许的蛋白的行为的可视化中的单元格。 Aip1p通常定位于肌动蛋白斑块在细胞内吞22的网站。量化Aip1p运动，超过50荧光Aip1p灶被跟踪在超过10个细胞中， 如图2A所示 ...

Watch this Scientific Journal Video about Aip1p Dynamics Are Altered by the R256H Mutation in Actin at JoVE.com

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

致病突变的肌动蛋白可以改变细胞骨架的功能。细胞骨架动力学是通过利用全内荧光显微镜荧光标记蛋白成像量化。作为一个例子，细胞骨架蛋白，Aip1p，已经改变了定位和运动在细胞中表达突变的肌动蛋白同种型，R256H。

Aip1p动力由R256H突变的肌动蛋白蚀变

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of ...

aip1p-dynamics-are-altered-by-the-r256h-mutation-in-actin

Research

JoVE Journal

Biology

7.9K 次观看.  University of Iowa. 致病突变的肌动蛋白可以改变细胞骨架的功能。细胞骨架动力学是通过利用全内荧光显微镜荧光标记蛋白成像量化。作为一个例子，细胞骨架蛋白，Aip1p，已经改变了定位和运动在细胞中表达突变的肌动蛋白同种型，R256H。 

视频: Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

表达GFP -肌动蛋白的内皮细胞的肌动蛋白细胞骨架的研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...