Name: cell lineage analyses and gene function studies using twin-spot marcm
Uploaded: 2017-03-02T14:00:00.000+00:00
Duration: 6 min 30 s
Description: Watch this Scientific Journal Video about Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM at JoVE.com

这项工作是由科学技术部的支持（MOST 104-2311-B-001-034）与细胞研究所和机体生物学，中央研究院，台湾。

1.建立使用所需转基因11，13 tsMARCM准备苍蝇<ol><li>生成tsMARCM准备苍蝇从被个别苍蝇进行11转基因的原始版本（ 见表1）。进行标飞遗传交叉方案中，其中先前已经描述的，通过将多个转基因在相同的飞股13。 <ol><li>在一种亲本系，组装FRT 40A，UAS - mCD8 :: GFP（第一报告，mCD8 :: GFP的转基因;转基因的表达是上游激活序列的启动子控制，其产生的膜-拴系记者下所述RNAi对大鼠簇0的表达;绿色荧光蛋白融合的分化8蛋白的小鼠群），和UAS-rCD2RNAi（抑制第二记者表达的抑制的˚F分化2，rcd2）在第 2 次染色体的左臂。地点 组织特异性-GAL4驱动在X或第三染色体，如果可能的话。 </li><li>在另一亲本系，组装FRT 40A，UAS-rCD2 :: RFP的转基因（第二记者，rCD2 :: RFP;另一膜-拴系的记者由具有红色荧光蛋白融合rCD2蛋白），和UAS-GFPRNAi （抑制mCD8 :: GFP表达的抑制;针对GFP表达的RNA干扰）的2 次染色体的左臂。放置热休克（HS）-FLP或组织specific- FLP在X或第三染色体，如果可能的话。 </li></ol></li><li>生成tsMARCM就绪苍蝇从由个人蝇14进行转基因的新版本（ 见表1）。进行标飞的遗传交叉方案中，已经描述previously，通过将多个转基因在同一飞股票13。 <ol><li>在一个亲本线，组装FRT位点和UAS-mCD8.GFP.UAS-rCD2i（mCD8 :: GFP和抑制rCD2表达:: RFP抑制器的组合转基因）的转基因一起的同一手臂第2 次或第 3 次的染色体（FRT和UAS-mCD8.GFP.UAS-rCD2i的转基因的适当对表1中所示）。放置组织特异性-GAL4司机比所选择的FRT位点的染色体，其他可能的话染色体。 </li><li>在另一亲本系，组装FRT位点和UAS-rCD2.RFP.UAS-GFPi（rCD2的其他组合的转基因:: RFP和抑制mCD8 :: GFP表达的抑制）在同一臂的转基因第2 次或第 3号染色体（适当对FRT的转基因的</青霉&gt;和UAS-rCD2.RFP.UAS-GFPi在表1中所示）。代替HS-FLP或大于所选择的FRT位点的染色体，其他可能的话染色体组织特异性-FLP。 </li></ol></li></ol> 2.跨tsMARCM准备飞往生成tsMARCM无性系后代<ol><li>跨tsMARCM就绪苍蝇（ 如，放10-15雄蝇从步骤1.1.1或1.2.1和20-30处女女步骤1.1.2或1.2.2一起飞）在飞食品小瓶新鲜对小瓶的壁订做酵母膏。 </li><li>保持在25℃的交叉tsMARCM就绪蝇2天收集受精卵之前提高交配的机会。 </li><li>频繁交叉tsMARCM就绪蝇转移到新鲜yeasted小瓶避免拥挤（点击向下的苍蝇或使用二氧化碳麻醉苍蝇便于转移;通过保持不超过80-100受精卵在一个10毫升飞食品L为了避免过多的孵幼虫）。 </li><li>培养tsMARCM动物（ 即，受精卵; tsMARCM动物的基因型的一个例子，如在图3中使用的，是HS-FLP [22]，w / w的; UAS-mCD8 :: GFP，UAS-rCD2RNAi，FRT 40A ，GAL4-GH146 / UAS-rCD2 :: RFP，UAS-GFPRNAi，FRT 40A + +）已在25℃下被放置在串行传送小瓶直到他们开发到所需阶段（ 例如，胚胎，幼体，或蛹）。 </li><li>放置包含tsMARCM动物在相同发育阶段到37℃水浴10，20，30，40所传送的小瓶中，50分钟，以确定热休克的最佳时间诱导FLP的表达，以产生感兴趣tsMARCM克隆。如果跳过这一步在tsMARCM实验中使用组织specific- FLP（HS-FLP是首选的神经系分析;对于这种偏好的原因先前已被描述15）。 注:使用在未来tsMARCM实验热休克的最佳时间，如果感兴趣的更多tsMARCM克隆想重复步骤2.5;通常，更多的FLP表达在诱导HS-FLP [22]相比于HS-FLP [1]具有相同的热休克的时间。 </li><li>文化在25℃的热震惊tsMARCM动物，直到他们达到合适的发育阶段，然后进行第3步。 </li></ol> 3.准备，着色漆，和粉煤灰山含脑克隆tsMARCM <ol><li>准备钳保护的菜肴。混合部分A（30克）和有机硅弹性体套件，带活性炭（0.25克）B部分（3克）。将混合物倒入合适的菜肴。 </li><li>解剖幼虫，蛹，或成人的脑袋上使用通过解剖显微镜观察的镊子保护盘钳在1×磷酸盐缓冲盐水的tsMARCM动物（PBS）中的。注:为飞大脑解剖协议已经说明此前16 ribed。 </li><li>固定在玻璃斑点板用含有1×PBS中的4％甲醛在室温下20分钟的飞大脑。处理在该玻璃斑点板为以下步骤剩余的飞大脑。 </li><li>冲洗苍蝇大脑中（含1×PBS中1％的Triton X-100）1％PBT，每洗三次，每次30分钟，以1％的PBT（漂洗三次:删除PBT和快速添加新PBT;清洗:除去PBT，添加新的PBT和孵化在新的PBT使用定轨振荡器）飞大脑。 </li><li>孵育初级抗体的混合物飞大脑在4℃或在室温下4小时过夜。初级抗体的混合物的一个例子是大鼠抗CD8抗体（1:100）:含有与小鼠抗Bruchpilot（BRP），以1％的PBT抗体（1:50），兔抗DsRed的抗体（800 1） 5％正常山羊血清（NGS）。 </li><li>冲洗飞大脑，用1％的PBT三次，然后把它们洗干净三次，每次30分钟，用1％的PBT。 </li><lI&gt;孵育次级抗体的混合物飞大脑在4℃或在室温下4小时过夜。二次抗体的混合物的一个例子是与绿色荧光染料（1:800）缀合的山羊抗大鼠IgG抗体，山羊抗兔IgG抗体有黄色荧光染料（1:800）缀合，和山羊抗小鼠IgG抗体结合有远红色荧光染料（1:800）在含有5％NGS 1％的PBT。 </li><li>冲洗飞大脑，用1％的PBT三次，然后把它们洗干净三次，每次30分钟，用1％的PBT。它们安装在与抗淬火试剂的微滑动。戴上微型滑动微型玻璃盖覆盖和保护的苍蝇大脑。密封使用透明指甲油微玻璃盖和微滑动的边缘。存储这些装并密封在4℃飞大脑标本。 </li></ol> 4.取，处理和分析tsMARCM克隆的荧光图像<ol><li>捕捉tsMARCM克隆fr的荧光图像嗡使用选择的共聚焦显微镜系统在步骤3.8中创建的样品。 </li><li> tsMARCM共焦荧光图像的项目堆叠成二维，使用具有所选择的共焦显微镜系统提供的图像处理软件扁平的图像（ 见图3B - 3E，3G - 3J，3L - 30， 和3Q - 3T为扁平的例子共聚焦荧光图像）。 </li><li>计数使用适当的图像处理软件（在ImageJ的17 如在"细胞计数"功能tsMARCM克隆的多细胞-NB边的细胞数，参见图3E，3J，3O，和3T用于小区的例子神经元的体）。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

该议定书中的关键步骤步骤1.1.1，1.2.1，2.3，2.5和3.2是获得良好tsMARCM结果是至关重要的。不表达在神经祖细胞GAL4组织特异性-GAL4驱动是优选的步骤1.1.1和1.2.1。避免过度拥挤在步骤2.3在飞食品小瓶生长的动物。因为感应延迟，FLP的在热休克的表达水平，和神经祖细胞（ 即，NBS和系膜）的平均分割时间是未知的，这是最好的，以改变热休克的时间（10，20，30 ，40，和50...

脑，由广大多样类型的神经元的，赋予动物感知，处理和来自外部世界的挑战的反应能力。成年果蝇中央脑的神经元被从神经干细胞的数量有限的派生，称为神经母细胞（NBS），开发1,2中。大多数国家统计局参加在果蝇脑内神经发生不对称分裂产生自我更新国家统计局和神经节母细胞（系膜细胞）和系膜细胞然后再通过另一轮分裂产生分化成神经元3两个子细胞（ 图1A ）。由于神经元形态的复杂性，并确定具体的神经元，正面镶嵌标签技术，以阻遏细胞标记物（MARCM）马赛克分析相关的挑战，被发明使visualizat一个单一的神经元即神经元的一小部分出来的周边，未标记的神经元4的人口的离子。 MARCM利用翻转酶（FLP）/ FLP识别目标（FRT）系统介导携带阻抑基因，其表达通常抑制报道基因4的表达的杂合等位基因的分割前体细胞内的同源染色体之间的位点特异性重组。的有丝分裂后，重组染色体分成双细胞，使得一个单元包含阻抑基因的纯合等位基因，而另一个单元没有阻遏基因的记者的在该小区（和其后代）表现不再4受阻。三个克隆型态通常在MARCM实验中发现，当FLP在的NB或系膜是随机诱导:单细胞和双小区GMC克隆，其描绘在单细胞resolutio神经元形态n和多细胞-NB克隆，它揭示了从一个共同的NB（ 图1B）衍生的神经元的整个形态模式。该MARCM技术已在果蝇的神经生物学研究中得到了广泛应用，包括神经类型识别用于重建大脑宽布线网络，神经系分析了披露神经元，参与细胞命运规范基因功能表型特征的发展历史，以及神经元形态发生和分化研究5，6，7，8，9，10。由于传统MARCM只标注诱导有丝分裂重组事件发生后两个子细胞（和系）中的一个，从侧无人盯防的潜在有用的信息丢失。这种限制妨碍了基本M的应用该切换细胞命运在快节奏的许多神经谱系的ARCM系统高分辨率分析或精密在不同动物11,12相同的神经元基因功能分析。 双点MARCM（tsMARCM）是一种先进的系统，标签从共同祖衍生用两种不同颜色的神经元，从而使有用的信息从双细胞两侧的恢复，从而克服了原有MARCM系统11的限制（ 图2A - 2C）。在tsMARCM系统中，两个RNA干扰（RNAi）为主抑制器是位于前体细胞内的同源染色体的反式位点，并且这些抑制剂的表达独立地抑制它们各自的记者（ 图2B）的表达。继通过FLP / FRT系统介导的位点特异性有丝分裂重组，在TWø基于RNAi的抑制成为分隔成两张细胞，以允许不同的记者（ 图2B）中的表达。两个克隆的图案，与多细胞-NB克隆相关的单细胞克隆和两个小区用GMC相关联的单细胞，通常见于一个tsMARCM实验（ 图2C）。信息从可利用作为另一侧上的参考双子细胞的一侧导出，使得到的高分辨率神经谱系分析，如出生约会标记的神经元，和用于精确调查在不同的动物相同的神经元表型分析神经基因功能11,12。这里，我们提出一个步骤一步协议描述如何在果蝇进行tsMARCM实验，其可以通过其他实验室使用，以扩大其神经发育的研究（以及其它组织的发展，如适用）。

<table><tbody><tr><td>Carbon dioxide (CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Local vendor</td><td>n.a.</td><td>Anesthetize flies</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pad</td><td>Local vendor</td><td>n.a.</td><td>Sort, cross and transfer flies</td></tr><tr><td>10x PBS pH 7.4</td><td>Uniregion Bio-Tech (or other vendors)</td><td>PBS001</td><td>Dissect, rinse, wash and immunostain fly brains</td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>252549</td><td>Fix fly brains</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Rinse, wash and immunostain fly brains</td></tr><tr><td>Normal goat</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>005-000-121</td><td>Immunostain fly brains</td></tr><tr><td>serum</td><td></td><td></td><td></td></tr><tr><td>Rat anti-mouse CD8 antibody</td><td>Invitrogen</td><td>MCD0800</td><td>Immunostain fly brains</td></tr><tr><td>Rabbit anti-DsRed antibody</td><td>Clonetech</td><td>632496</td><td>Immunostain fly brains</td></tr><tr><td>Mouse anti-Brp antibody</td><td>Developmental Studies Hybridoma Bank</td><td>nc82</td><td>Immunostain fly brains</td></tr><tr><td>Goat anti-rat IgG antibody conjugated with Alexa Fluor 488</td><td>Invitrogen</td><td>A11006</td><td>Immunostain fly brains</td></tr><tr><td>Goat anti-rabbit IgG antibody conjugated with Alexa Fluor 546</td><td>Invitrogen</td><td>A11035</td><td>Immunostain fly brains</td></tr><tr><td>Goat anti-mouse IgG antibody conjugated with Alexa Fluor 647</td><td>Invitrogen</td><td>A21236</td><td>Immunostain fly brains</td></tr><tr><td>SlowFade gold antifade reagent</td><td>Molecular Probes</td><td>S36936</td><td>Mount fly brains and protect quenching of fluorescence&amp;nbsp;</td></tr><tr><td>PYREX 9 depression glass spot plate</td><td>Corning</td><td>7220-85</td><td>Dissect, rinse, wash and immunostain fly brains</td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>World Precision Instruments</td><td>SYLG184</td><td>Make black Sylgard dishes to protect forceps during brain dissection</td></tr><tr><td>Activated charcoal</td><td>Sigma-Aldrich</td><td>242276-250G</td><td>Make black Sylgard dishes</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11252-30</td><td>Dissect and mount fly brains</td></tr><tr><td>Micro slide</td><td>Corning</td><td>2948-75x25</td><td>Mount fly brains</td></tr><tr><td>Micro cover glass No. 1.5</td><td>VWR International</td><td>48366-205</td><td>Mount fly brains</td></tr><tr><td>Nail polish</td><td>Local vendor</td><td>not available</td><td>Seal micro cover glass on micro slides</td></tr><tr><td>Incubator&amp;nbsp;</td><td>Kansin Instruments</td><td>LTI603</td><td>culture flies at 25 &amp;deg;C</td></tr><tr><td>(or other vendors)</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td>Kansin Instruments</td><td>WB212-B2</td><td>Induce heat-shock in flies at 37 &amp;deg;C</td></tr><tr><td>(or other vendors)</td><td></td><td></td><td></td></tr><tr><td>Orbital shaker</td><td>Kansin Instruments</td><td>OS701</td><td>Wash and immunostain fly brains</td></tr><tr><td>(or other vendors)</td><td></td><td></td><td></td></tr><tr><td>Dissection microscope</td><td>Leica</td><td>EZ4</td><td>Sort, cross and transfer flies; Dissect, rinse, wash and immunostain fly brains</td></tr><tr><td>Confocal microscope</td><td>Zeiss (or other vendors)</td><td>LSM 700 (or other models)</td><td>Image tsMARCM clones</td></tr><tr><td>image-processing software 1&lt;br/&gt; (&lt;em&gt;e.g.&lt;/em&gt;, Zeiss LSM image browser)&amp;nbsp;</td><td>Zeiss</td><td>not available</td><td>Project stacks of confocal images</td></tr><tr><td>image-processing software 2&lt;br/&gt; (&lt;em&gt;e.g.&lt;/em&gt;, ImageJ)&amp;nbsp;</td><td>not available</td><td>not available</td><td>Count cell number of tsMARCM clones</td></tr></tbody></table>

cell lineage analyses and gene function studies using twin-spot marcm

中号 osaic带有R epressible 有丝米 arker 一 nalysis（MARCM）是一种已被广泛应用在果蝇的神经生物学研究来描绘复杂的形态和内，否则无人盯防和泰然自若操纵基因功能的神经元亚群的积极镶嵌标签制度生物。在MARCM系统中产生遗传马赛克通过同源染色体之间的位点特异性重组内分裂前体细胞的有丝分裂过程中，以产生两个标记（MARCM克隆）和未标记的子细胞介导的。所述MARCM方法的扩展，称为双点MARCM（tsMARCM），标签都从具有两种不同颜色的共同祖先来源的双细胞。这种技术被开发，以使从两个半谱系有​​用信息的检索。通过综合分析对不同tsMARCM克隆的tsMARCM系统允许高分辨率神经系mapping至揭示从共同祖细胞产生的标记的神经元的确切出生顺序。此外，tsMARCM系统还通过允许不同动物的相同神经元的表型分析延伸基因功能的研究。在这里，我们将介绍如何应用tsMARCM系统，方便神经发育研究果蝇 。

该tsMARCM系统已被用于通过检索来自共同的NB衍生的神经元的重要信息，以促进神经谱系分析和基因功能研究。该系统已被用于识别大部分（如果不是全部）的神经元类型，确定每个神经元类型的细胞数量，并确定使用tsMARCM这些神经元的出生顺序11，12，18（见讨论部分的详细说明开展神经系...

Watch this Scientific Journal Video about Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM at JoVE.com

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

这里，我们提出了一个协议，用于一个马赛克标记技术，它允许从一个共同的祖细胞在两种不同颜色来源的神经元的可视化。这有利于与出生约会单个神经元中不同个体的相同的神经元的能力，研究基因功能的神经谱系分析。

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 ...

cell-lineage-analyses-and-gene-function-studies-using-twin-spot-marcm

Research

JoVE Journal

Developmental Biology

9.9K 次观看.  Academia Sinica. 这里，我们提出了一个协议，用于一个马赛克标记技术，它允许从一个共同的祖细胞在两种不同颜色来源的神经元的可视化。这有利于与出生约会单个神经元中不同个体的相同的神经元的能力，研究基因功能的神经谱系分析。 

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

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 ...

发育生物学

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

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 ...

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

公司重点调查的社区图书馆筛选确定关键 CTCF 边界

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

遗传学

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

生物化学

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 ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

用于单细胞测序的原肠胚胚分离和基因分型

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

作者聚焦：以单细胞分辨率分析谱系特异性突变胚胎的管道

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

细胞谱系分析和基因功能研究使用双点MARCM

摘要

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方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

细胞系标记的共定位和番茄信号

公司重点调查的社区图书馆筛选确定关键 CTCF 边界

单分子鱼的转录活性等位基因单细胞分析

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

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束