Name: cell lineage analyses and gene function studies using twin-spot marcm
Uploaded: 2017-03-02T14:00:00
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...

<ol>
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S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+dissecting+Drosophila+melanogaster+brains+for+live+imaging+or+immunostaining.">A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining.</a> Nat Protoc. 1 (4), 2110-2115 (2006).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Lin, S., Kao, C. F., Yu, H. H., Huang, Y., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+analysis+of+Drosophila+lateral+antennal+lobe+neurons+reveals+notch-dependent+binary+temporal+fate+decisions.">Lineage analysis of Drosophila lateral antennal lobe neurons reveals notch-dependent binary temporal fate decisions.</a> PLoS Biol. 10 (11), e1001425 (2012).</li><li>Zhan, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Dscam+diversity+in+regulating+axon+guidance+in+Drosophila+mushroom+bodies.">Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies.</a> Neuron. 43 (5), 673-686 (2004).</li><li>Griffin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+twin+spot+generator+for+differential+Drosophila+lineage+analysis.">The twin spot generator for differential Drosophila lineage analysis.</a> Nat Methods. 6 (8), 600-602 (2009).</li><li>Potter, C. J., Tasic, B., Russler, E. V., Liang, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Q+system:+a+repressible+binary+system+for+transgene+expression,+lineage+tracing,+and+mosaic+analysis.">The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.</a> Cell. 141 (3), 536-548 (2010).</li><li>Lai, S. L., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mosaic+with+dual+binary+transcriptional+systems+in+Drosophila.">Genetic mosaic with dual binary transcriptional systems in Drosophila.</a> Nat Neurosci. 9 (5), 703-709 (2006).</li><li>Kao, C. F., Yu, H. H., He, Y., Kao, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carbon dioxide (CO2)</td>
			<td>Local vendor</td>
			<td>n.a.</td>
			<td>Anesthetize flies</td>
		</tr>
		<tr>
			<td>CO2 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 rowspan="2">Jackson ImmunoResearch&#160;</td>
			<td rowspan="2">005-000-121</td>
			<td rowspan="2">Immunostain fly brains</td>
		</tr>
		<tr>
			<td>serum</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&#160;</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 rowspan="2">Incubator&#160;</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">LTI603</td>
			<td rowspan="2">culture flies at 25&#176;C</td>
		</tr>
		<tr>
			<td>(or other vendors)</td>
		</tr>
		<tr>
			<td rowspan="2">Water bath</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">WB212-B2</td>
			<td rowspan="2">Induce heat-shock in flies at 37&#176;C</td>
		</tr>
		<tr>
			<td>(or other vendors)</td>
		</tr>
		<tr>
			<td rowspan="2">Orbital shaker</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">OS701</td>
			<td rowspan="2">Wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>(or other vendors)</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 
			(e.g., Zeiss LSM image browser)&#160;</td>
			<td>Zeiss</td>
			<td>not available</td>
			<td>Project stacks of confocal images</td>
		</tr>
		<tr>
			<td>image-processing software 2 
			(e.g., Image-J)&#160;</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 ...

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

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

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 ...

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

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 ...

Co-localization of Cell Lineage Markers and the Tomato Signal

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 ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

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

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

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 ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Live Cell Imaging of Chromosome Segregation During Mitosis

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

Tracing Gene Expression Through Detection of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-galactosidase Activity in Whole Mouse Embryos

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...