Name: laser microdissection applied to gene expression profiling of subset of cells from the drosophila wing disc
Uploaded: 2010-04-30T17:00:00
Description: Watch this Scientific Journal Video about Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc at JoVE.com

作者感谢教授。卢嘉勒康帕内拉和AMRA中心的能力，那不勒斯的费德里科第二大学，那不勒斯，意大利，为他们提供与使用激光microdissector，议员及文森佐Vicidomini在三维动画的慷慨帮助。

第1部分。制备受到具体的和本地化的RNAi 的果蝇成虫翼光盘。 作为生物材料中，我们使用受到GAL4/UAS 系统本地化和特定的基因沉默的转基因幼虫获得的果蝇成虫翼光盘。这幼虫的后代orginates从一个涉及到两个亲本的遗传交叉：携带GAL4的驱动器，第二的UAS响应。除了表达GAL4在一个特定的时间和/或空间格局，车手阵容进行UAS - GFP的记者说，允许GAL4表达域的?...

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	<li>Elliott, D. A., Brand, A. H., Dahmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GAL4+System+A+Versatile+System+for+the+Expression+of+Gene.">The GAL4 System A Versatile System for the Expression of Gene.</a> , (2008).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.+.">Wing disc development in the fly: the early stages. .</a> Current Opinion in Genetics &#38; Development. 11, 470-475 (2001).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-U1-151-U1 (2007).</li><li>Martin, F. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+the+control+of+growth+in+the+Drosophila+wing+imaginal+disc.">Compartments and the control of growth in the Drosophila wing imaginal disc.</a> Development. 133, 4421-4426 (2006).</li><li>Tabata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+morphogen+gradients.">Genetics of morphogen gradients.</a> Nature Reviews Genetics. 2, 620-630 (2001).</li><li>Moreno, E. &. a. m. p. ;. a. m. p., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dMyc+transforms+cells+into+super-competitors.">dMyc transforms cells into super-competitors.</a> Cell. 117, 117-129 (2004).</li></ol>

尊重unsilenced翼光盘车厢前/后所取得的基础表达水平降低到40％的沉默后，对选定的基因X的活性被发现。相比之下，其公认的目标（基因Ÿ）被发现显着上调（7折的增加），导致我们要验证的假说，它是基因X.负调控我们得出结论，上述描述的实验方法可以成功地应用到不同的调控途径的假定目标的验证。 此外，我们推测，在改善RNA提取的产量可以迅速使...

飞株果蝇无人机沉默线可从VDRC RNAi的收集2。 GAL4驱动线的集合，可在布卢明顿果蝇联合中心（美国印第安那州）。 设备所需设备，包括激光microdissector（莱卡LMD6000），库尔特离心22R（贝克曼），PCR（BIO - RAD我的扩增仪），实时定量PCR（BIO - RAD iQ5）。 工具所需的工具包括：显微切割钳，刷子，玻璃井，悬滴幻灯片，注射器针头，与PET膜，塑料管（50毫升，05毫升，0.25毫升）的金属框架上的昆虫引脚。

laser microdissection applied to gene expression profiling of subset of cells from the drosophila wing disc

异质性的组织已被证明是一个限制因素，可以从生物样品中产生的信息量，影响下游分析。考虑到复杂和动态的许多组织内现有的蜂窝协会，以总结在体内的相互作用深入的分子分析，必须在家乡范围内能够分析特定的细胞群。激光介导显微切割可以达到这个目标，可以明确识别和细胞，直接镜检可视化下的利益收获成功的同时保持分子的完整性。我们已应用此技术来分析定义的区域内发展的果蝇翅膀的光盘，它代表一个有利的模型系统，研究增长的控制，细胞分化和器官的基因表达。幼虫成虫光盘早熟分为前，后，背，腹车厢谱系限制边界。利用诱导GAL4 - UAS的二元表达系统，每个车厢可以具体表达适当GAL4驱动构造控制下的UAS - GFP的转基因在转基因果蝇标记。在转基因光盘，离散细胞亚群的基因表达分析可以精确地确定后介导的激光显微切割，使用荧光GFP信号引导激光切割。 各种下游应用当中，我们集中在RNA转录分析后，本地化的RNA干扰（RNAi）。随着RNAi技术的出现，绿色荧光蛋白标记可以再加上本地化的一个特定基因的敲除，使确定的特定的基因沉默的一个独立的细胞群的转录反应。为了验证这种方法，我们解剖后（绿色荧光蛋白表达标记）光盘相当于地区，和前（不知名）后，在一个广泛表达的基因P舱的区域沉默的车厢。 RNA提取从显微切割沉默和unsilenced地区和比较基因表达分析实时定量RT - PCR检测确定。我们表明，该方法可以有效地应用于果蝇成虫光盘内的细胞亚群的准确转录。事实上，大量的光盘准备RNA的来源，而一般假定细胞的同质性，这是众所周知的转录表达，可以在这些结构有很大不同，在位置信息的后果。使用本地化的荧光GFP信号引导激光切割，可进行更准确的转录分析和盈利应用于不同的应用程序，包括在其本土范围内的谈话内容截然不同的细胞谱系分析。

Watch this Scientific Journal Video about Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc at JoVE.com

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

激光显微切割技术应用于基因表达谱分析果蝇翼光盘的具体车厢，本地化的RNAi<em&gt;在体内</em&gt;。从沉默和unsilenced车厢相当于地区提取的RNA定量RT - PCR分析，以确定在本土组织微生态的背景下比较的基因表达图谱。

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

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Biology

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Capture Microdissection of Mammalian Tissue

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

科研

生物学

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The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

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标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

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全球合成的RNA在单细胞水平以下的低氧分析

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Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...