Name: co-localization of cell lineage markers and the tomato signal
Uploaded: 2016-12-28T14:00:00.000+00:00
Duration: 10 min 56 s
Description: Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

This study was supported by NIH grant DE025014 to JQF.

所有协议进行审查和机构动物护理和使用委员会（IACUC）在得克萨斯A＆牙科M大学学院批准。 1.动物育种<ol><li>在这项研究中使用三种动物模型。为了研究胚胎软骨细胞的形成髁的命运，第一次使用COL10A1 Cre重组 15只和ROSA26 tdTomato越过他们（B6; 129S6- 亿吨（ROSA）26Sor TM9（CAG-tdTomato）HZE / J）小鼠获得Col10a1-综合招聘考试及ROSA26 tdTomato老鼠。接下来，跨越这些小鼠2.3Col1a1-GFP小鼠16。使用COL10A1 Cre重组; ROSA26 tdTomato; 2.3Col1a1-GFP的小鼠进行细胞谱系追踪试验（ 图1B）。 </li><li>为了研究产后软骨细胞的命运，遵循相同的程序与步骤1.1，但使用蛋白多糖，CRE ERT2（ 总比分Cre重组ERT2）17,18线，并保持此Agg Cre重组ERT2; ROSA26 tdTomato; 2.3Col1a1-GFP的小鼠进行细胞谱系追踪试验（ 图1C）。注入14日龄他莫昔芬。 </li><li>要结合细胞谱系追踪与免疫荧光技术，跨AGG的Cre重组ERT2小鼠和ROSA26 tdTomato老鼠。使用该实验中携带两个基因小鼠和3日龄（ 图1D）注入他莫昔芬。 </li></ol> 2.材料准备<ol><li>溶解在10％的乙醇和90％的玉米油的他莫昔芬粉末以10mg / ml的浓度。用于注射的剂量为75毫克/公斤。 </li><li>溶解在PBS中的多聚甲醛（PFA）粉末，以4％的浓度，用2M的氢氧化钠以调节pH值至7.4。使用4％PFA解决方案，以修复牺牲后，组织（下颌骨髁状突和后腿这里使用）。由于其毒性，在手套和口罩罩处理PFA。 </li><li>溶解在蒸馏水中的EDTA粉末的浓度为10％，用2M的氢氧化钠以调节pH值至7.4。使用10％的EDTA脱钙髁突及后腿。 </li><li>溶解在15％和30％的浓度在PBS中蔗糖的粉末。使用蔗糖溶液脱钙后脱水的组织。 </li><li>溶解在PBS中的透明质酸粉末以2毫克/毫升，pH 5.0的浓度。这个解决方案是免疫抗原修复。 </li><li>用1.5毫升管以制备含有3％牛血清白蛋白（BSA）和20％山羊血清的PBS Runx2的或DMP1免疫荧光染色的封闭溶液。 </li><li>用1.5毫升管以制备包含2％山羊血清的PBS Runx2的或DMP1免疫荧光染色的初级抗体溶液。第一抗体的浓度为1:400为Runx2的和1:100 DMP1。 </li><li>用1.5毫升管以制备兔IgG溶液作为对照用于免疫荧光染色，以避免假阳性结果。该溶液含有在PBS中2％山羊血清。的兔IgG为Runx2的控制的浓度为1:400;为DMP1，1:100。同时进行实验组和对照染色。 </li><li>使用1.5ml的管以制备包含2％山羊血清的PBS Runx2的或DMP1免疫荧光染色的二级抗体溶液。使用的第二抗体以1稀释:500。 </li></ol> 3.将准备共聚焦显微镜（细胞系只有跟踪，用免疫无组合） <ol><li>在有利的时间点注入在此Agg-CRE ERT2小鼠他莫昔芬。 <ol><li>首先，从笼子里取出一只老鼠。然后，用左手拇指和食指抓住鼠标的背部皮肤，翻过来，露出腹部。用右手按住注射器。最佳入口点注射是在LEFT或下腹部的右侧，避免了肝脏和膀胱。 </li><li>保持平行注射器鼠标的后腿和腹膜内注射。用于注射的剂量为75毫克/公斤。分别为18克， - 小鼠在2周，3周，4周龄的年龄的加权数是约7 - 8克，11 - 13克，和16。 </li></ol></li><li>麻醉与赛拉嗪/ Ketaset组合的老鼠。 <ol><li>为了制备工作溶液，先用蒸馏水稀释甲苯噻嗪和Ketaset至1毫克/毫升和5毫克/ ml的浓度，分别。 2比:接着，在1混合甲苯噻嗪和Ketaset。用于注射的剂量为30微升/ g以下。 </li><li>注入如在步骤3.1。按捏鼠的脚踝确认麻醉。鼠标是无意识的，如果它没有反应。 </li></ol></li><li>麻醉后灌注用4％PFA的小鼠。 <ol><li>鼠标失去知觉后，固定在板上鼠标的四条腿entirelŸ露出腹部。 </li><li>饱和，用70％的乙醇的腹部并进行切除从下腹部到沿中间线颈部。捏和同时皮肤拉向侧面以显示腹膜。用剪刀清扫，使纵向切除。 </li><li>切断并去掉前面的肋骨暴露心脏。从一个22克的注射器左心室穿刺心脏，持注射器，同时切槽的右心耳。 </li><li>慢慢注入4％PFA，其沿着心血管系统灌注而潮红出从右侧耳廓的切割。在PFA的灌注体积为1毫升/克。执行在I级生物安全柜是难以输送到大楼的排气系统这一步。 </li></ol></li><li>剥离鼠标的皮肤，并把整个身体成含有40毫升的4％的PFA在4℃固定过夜的50ml聚丙烯离心管中。 </li><li>使用解剖剪刀和＃3，＃5镊子小心地清除体内的下颌骨和后腿，并除去表面的肌肉和肌腱。执行在I级生物安全柜是难以输送到大楼的排气系统这一步。 </li><li>切下颌骨成两片在第三臼齿的远侧区域。同样，切股骨和胫骨中的中段，以暴露骨髓腔，以加速脱钙。把包括所述髁和髁突的一部分，并与后腿成40毫升10％的EDTA一起在4℃下进行2脱钙 - 4天50ml的聚丙烯离心管中。 </li><li>用50毫升15％的蔗糖在50ml的聚丙烯离心管中，以脱水髁和后腿过夜，在4℃。 </li><li>使用30％的蔗糖在50ml的聚丙烯离心管中，以脱水髁和后腿在4℃过夜。 </li><li>沿矢状面，嵌入与样品华侨城在冷冻切片机切板。 <ol><li>水平放置髁或在安装模具的后腿。淹没组织OCT和它留在冷冻切片机，直到华侨城冻结。 </li><li>安装在剪板机华侨城块。切割，以确保在OCT是完全冻结之前等待约15分钟。 </li></ol></li><li>切髁和后腿成10微米的部分。收集幻灯片和商店的部分在-20°C。 </li><li>孵育在37℃室中的载片染色之前以除去水。 </li><li>用蒸馏水洗涤载玻片两次5分钟。 </li><li>擦去水绕每个部分。使用疏水屏障用笔圈出部分拖放DAPI或无荧光防褪色的安装解决方案进入这个圈子。小心放下盖玻片。 </li></ol> 4.免疫组织化学染色为Runx2的和DMP1 注:IgG抗体CON控制是必要的免疫组化染色，避免假阳性信号。需要同时执行对实验组和对照组的染色。 <ol><li>孵育在37℃室中的载玻片染色之前以除去水。 </li><li>用蒸馏水冲洗幻灯片的两倍。 </li><li>使用疏水屏障笔来圈上滑动的所有部分。从该步骤中，添加所有制备的溶液的成圈以完全覆盖部分。 </li><li>治疗与透明质酸酶的部分在潮湿室中在37℃下进行30分钟。的溶液（在步骤4.5 - 4.8）的体积取决于该部分的大小。用于髁50微升溶液和100微升的长骨。用PBST（PBS中含有0.1％吐温20）三次清洗。 </li><li>制备和应用封闭溶液到每一个部分，并培育它们在潮湿室中在室温下1小时。 </li><li>孵育主要章节在4℃过夜抗体溶液（兔抗小鼠Runx2的，或兔抗小鼠DMP1）。用PBS三次洗涤。 </li><li>孵育二级抗体溶液（山羊抗兔，的Alexa Fluor 488），用于在室温下2小时的部分。用PBS三次洗涤。 </li><li>擦掉水绕的部分拖放DAPI到了中圈，以支付幻灯片上的部分。小心放下盖玻片。 </li></ol> 5.共聚焦显微镜<ol><li>在波长范围从488微米（绿色），561微米（红色）捕获利用共聚焦显微镜的荧光细胞图像。在200Hz以多个堆叠图像19（1024×1024尺寸），使用10X，20X，63X和镜头。 </li></ol>

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The authors declare that they have no competing financial interests or other conflicts of interest.

由于技术限制，它始终是难以调查细胞在体内的行为。然而，细胞谱系追踪技术已被证明是用于研究细胞生物学7-9的有力工具。在这项研究中，我们进一步通过与免疫相结合的方法进行改进。以这种方式，细胞命运可以由多个相关的标志物，从而拓宽谱系追踪中的应用来定义。此外，免疫荧光和番茄信号的此共定位同时显示的创始人细胞，它们的位置，以及它们的分化状态的后代?...

在开发过程中，软骨内骨形成占骨架体积的80％以上。人们普遍认为，这首先肥大软骨细胞的凋亡。接着，从底层骨髓细胞侵入并启动血管生成，随后通过骨髓和骨膜衍生细胞-1,2-新骨沉积。肥大软骨细胞的细胞命运（HCS），然而，一直是人们争论的一个问题，几十年来3。最初，碳氢化合物被认为是软骨细胞分化途径的端部，和凋亡普遍认为是碳氢化合物的最终命运。现在，一些研究人员认为，至少有一些碳氢化合物能够生存，并有助于软骨内成骨。虽然他们提议生长板软骨不得不转分化成基于超微结构，免疫组织化学染色的成骨细胞的能力，以及在体外研究46中 ，这些方法瓦特埃雷在证明对成骨细胞谱系的贡献软骨明确。 该细胞谱系追踪技术提供了一种更严格的方式来研究细胞命运。简要地说，一个重组酶，这是只在特定类型的细胞中表达，刺激报道基因的表达。以这种方式，这种类型的细胞及其后代被永久标记7。该Cre的loxP系统在谱系追踪常用。酶Cre（重组酶酶）将切除两个loxP位点之间的停止序列和激活报告在特定的细胞系（ 图1A）。在一些情况下，研究者可以选择有利的时间点，通过使用一种药物，例如他莫昔芬激活酶Cre，引起酶Cre熔合到雌激素受体（酶Cre ERT2）8的改进形式。荧光记者已成为谱系追踪实验，因为他们大幅降低复杂性的标准和提高细胞命运跟踪8,9的准确性和效率。 tdTomato是变荧光报告之间的最佳选择，因为它具有最亮的荧光蛋白和最强的萤光，使它很容易地可视化7（ 图1A）。 通过使用ROSA26 tdTomato谱系追踪系统，我们组和其他研究者已经证明，碳氧化合物可以在开发过程中10-14改变其表型成骨细胞。要做到这一点，我们开发了两套与ROSA26 tdtomato（普遍存在的表达在所有的细胞）/ Cre的（特定于软骨细胞）的小鼠的跟踪组合:2.3Col1a1-GFP（特定于成骨细胞）和免疫荧光（特定于骨细胞）。该数据表明，这两种方法都是可行的方法，来研究细胞的命运体内 。

<table><tbody><tr><td>Tamoxifen&amp;nbsp;</td><td>Sigma</td><td>T5648</td><td>activate the Cre event&amp;nbsp;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma&amp;nbsp;</td><td>P6148</td><td>fix the sample</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Alfa Aesar</td><td>A10713</td><td>decalcify the hard tissue</td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma</td><td>S0389</td><td>dehydrate the tissue</td></tr><tr><td>Hyaluronidase from bovine testes&amp;nbsp;</td><td>Sigma</td><td>H4272</td><td>retrieve antigen for immunochemical staining</td></tr><tr><td>Bovine serum albumin</td><td>Sigma&amp;nbsp;</td><td>A3059</td><td>blocking solution&amp;nbsp;</td></tr><tr><td>primary antibody for Runx2&amp;nbsp;</td><td>Cell Signal</td><td>D1L7F</td><td>primary antibody for immunochemical staining</td></tr><tr><td>primary antibody for DMP1</td><td>provided by Dr. Chunlin Qin</td><td></td><td>primary antibody for immunochemical staining</td></tr><tr><td>anti-rabbit IgG</td><td>Sigma</td><td>18140</td><td>control for immunochemical staining</td></tr><tr><td>secondary antibody&amp;nbsp;</td><td>Invitrogen</td><td>A11008</td><td>second antibody for immunochemical staining</td></tr><tr><td>OCT</td><td>Tissue-Tek</td><td>4583</td><td>embed the sample for frozen section</td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337</td><td>PBST</td></tr><tr><td>non-fluorescing antifade mountant</td><td>Life technologies</td><td>P36934</td><td>mounting slides</td></tr><tr><td>DAPI</td><td>Life technologies</td><td>P36931</td><td>nuclear staining</td></tr><tr><td>Hydrophobic Barrier Pen</td><td>Vector Laboratories</td><td></td><td>circle the section on the slide for for immunochemical staining&amp;nbsp;</td></tr><tr><td>Xylazine</td><td>AnaSed</td><td></td><td>anesthetization&amp;nbsp;</td></tr><tr><td>Ketaset&amp;nbsp;</td><td>Zoetis</td><td></td><td>anesthetization&amp;nbsp;</td></tr><tr><td>cryosection machine</td><td>Leica</td><td>CM1860 UV</td><td></td></tr><tr><td>confocal microscope</td><td>Leica</td><td>DM6000 CFS&amp;nbsp;</td><td></td></tr></tbody></table>

co-localization of cell lineage markers and the tomato signal

细胞谱系追踪系统已在发育生物学研究中主要使用。利用Cre重组酶的允许记者在特定细胞系和所有后代的活化。在这里，我们使用的细胞谱系追踪技术来证明软骨细胞长骨以及使用2种酶Cre，COL10A1 Cre重组和聚蛋白多糖-Cre重组ERT2（ 此Agg-Cre重组ERT2）的下颌骨髁发育过程中直接转变成成骨细胞和骨细胞，越过与ROSA26 tdTomato。无论Col10和蛋白聚糖被公认的标记物软骨细胞。 在此基础上，我们开发了一种新方法，细胞谱系通过分析细胞特异性标志物的表达在用荧光免疫-以限定细胞的命运一起跟踪。 RUNX2（对于早期骨细胞的标记）和牙本质基质蛋白1（DMP1;对于晚期骨细胞的标记）为用于鉴定软骨衍生骨细胞和它们的分化状态。这种组合不仅拓宽细胞谱系追踪中的应用，而且还简化了化合物的小鼠的产生。更重要的是，亲本细胞的后代的数量，位置和分化状态同时显示，提供比细胞谱系单独跟踪的详细信息。总之，细胞谱系追踪技术和免疫荧光共应用是用于体内研究细胞生物学的有力工具。

软骨细胞直接转变成在颌骨髁状突和长骨骨细胞（成骨细胞和骨细胞）。 聚集蛋白聚糖 ，对软骨的关键基因，主要表现为早期和成熟的软骨细胞18。其结果是，在2周的年龄在此Agg-Cre重组ERT2三苯氧胺的注射液; ROSA26 tdTomato激活小鼠红番茄记者在所有的软骨细胞和它们?...

Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

Co-localization of Cell Lineage Markers and the Tomato Signal

我们制定了两套在ROSA26 tdtomato（在所有细胞中广泛表达）/ Cre重组（特别是软骨细胞表达）小鼠跟踪组合:一个2.3Col1a1-GFP（具体以成骨细胞）和一个与免疫（具体以骨细胞）。该数据证明软骨细胞的直接转化成骨细胞。

细胞系标记的共定位和番茄信号

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

co-localization-of-cell-lineage-markers-and-the-tomato-signal

Research

JoVE Journal

Developmental Biology

12.1K 次观看.  Texas A&M University College of Dentistry. 我们制定了两套在ROSA26 tdtomato（在所有细胞中广泛表达）/ Cre重组（特别是软骨细胞表达）小鼠跟踪组合:一个2.3Col1a1-GFP（具体以成骨细胞）和一个与免疫（具体以骨细胞）。该数据证明软骨细胞的直接转化成骨细胞。 

视频: Co-localization of Cell Lineage Markers and the Tomato Signal

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

科研

发育生物学

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

细胞谱系分析和基因功能研究使用双点MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

不同植物种的射击顶端共聚焦活图

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

免疫与感染

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

生物学

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

神经科学

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

使用双标记 （MADM） 的马赛克分析开发脑皮质的血统跟踪和克隆分析

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...

细胞系标记的共定位和番茄信号

摘要

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此视频中的章节

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

细胞谱系分析和基因功能研究使用双点MARCM

不同植物种的射击顶端共聚焦活图

IL-22表达淋巴细胞使用记者小鼠的可视化

蛋白质中 Plasmodesmal 定位序列的识别在底中

原位杂交技术在植物中的精确定位成绩单

细胞特异性分析

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

使用双标记（MADM）的马赛克分析开发脑皮质的血统跟踪和克隆分析

细胞系标记的共定位和番茄信号

摘要

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此视频中的章节

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

细胞谱系分析和基因功能研究使用双点MARCM

不同植物种的射击顶端共聚焦活图

IL-22表达淋巴细胞使用记者小鼠的可视化

蛋白质中 Plasmodesmal 定位序列的识别在底中

原位杂交技术在植物中的精确定位成绩单

细胞特异性分析

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

使用双标记 （MADM） 的马赛克分析开发脑皮质的血统跟踪和克隆分析

使用双标记（MADM）的马赛克分析开发脑皮质的血统跟踪和克隆分析