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 border="0" cellpadding="0" cellspacing="0" width="1099">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tamoxifen&#160;</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:136px;">T5648</td>
			<td style="width:496px;">activate the Cre event&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td>fix the sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid</td>
			<td>Alfa Aesar</td>
			<td>A10713</td>
			<td>decalcify the hard tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose&#160;</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>dehydrate the tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyaluronidase from bovine testes&#160;</td>
			<td>Sigma</td>
			<td>H4272</td>
			<td>retrieve antigen for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Bovine serum albumin</td>
			<td>Sigma&#160;</td>
			<td>A3059</td>
			<td>blocking solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">primary antibody for Runx2&#160;</td>
			<td>Cell Signal</td>
			<td>D1L7F</td>
			<td>primary antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">primary antibody for DMP1</td>
			<td style="width:220px;">provided by Dr. Chunlin Qin</td>
			<td style="width:136px;"></td>
			<td>primary antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-rabbit IgG</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:136px;">18140</td>
			<td>control for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">secondary antibody&#160;</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>second antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>embed the sample for frozen section</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337</td>
			<td>PBST</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">non-fluorescing antifade mountant</td>
			<td>Life technologies</td>
			<td>P36934</td>
			<td>mounting slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Life technologies</td>
			<td>P36931</td>
			<td>nuclear staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrophobic Barrier Pen</td>
			<td>Vector Laboratories</td>
			<td></td>
			<td>circle the section on the slide for for immunochemical staining&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td style="width:220px;">AnaSed</td>
			<td></td>
			<td>anesthetization&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketaset&#160;</td>
			<td>Zoetis</td>
			<td></td>
			<td>anesthetization&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cryosection machine</td>
			<td style="width:220px;">Leica</td>
			<td style="width:136px;">CM1860 UV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">confocal microscope</td>
			<td style="width:220px;">Leica</td>
			<td>DM6000 CFS&#160;</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

Universidad de Cartagena UDEC

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

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

科研

发育生物学

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

在SH-SY5Y人神经母细胞瘤分化

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

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 ...

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 ...

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 ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...