Name: a high-throughput method to globally study the organelle morphology in s. cerevisiae
Uploaded: 2009-03-02T00:00:00
Description: Watch this Scientific Journal Video about A high-throughput method to globally study the organelle morphology in S. cerevisiae at JoVE.com

<p class="jove_content">这项工作是支持的生物技术和生物科学研究理事会（批31/C15982），加拿大卫生研究院，加拿大，不列颠哥伦比亚省知识发展基金，创新基金的迈克尔史密斯健康研究基金会（授予民事司法制度改革雄狮），并为视力扑灭。 </p>

<p class="jove_title">材料和方法： </p><ol><li>酵母菌株： <ul><li> ACP1 - GFP（绿色荧光蛋白：）：C -末端标记GFP的ACP1生成一个使用（包含GFP和<em>HIS5）</em>质粒pKT128 PCR介导的同源重组。共聚焦显微镜和菌落PCR确认阳性转化。应变背景BY7043（MAT阿尔法can1Δ：STE2pr lue2lyp1Δhis3Δ1leu2Δ0ura3Δ0met15Δ0）从布恩实验室（塘和Boone，2006年）。 </li><li>缺失突变体阵列（DMA）（XXX：：KanR）：它是一个非必需基因的缺失突变体约5000的数组。?...

<ol>
	<li>Amberg, D. C., Burke, D., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual</em>. , (2005).</li><li>Tong, A. H., Boone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+genetic+array+analysis+in+Saccharomyces+cerevisiae.">Synthetic genetic array analysis in Saccharomyces cerevisiae.</a> <em style='font-style: italic'>Methods Mol Biol</em>. <b>313</b>, 171-192 (2006).</li></ol>

<p class="jove_content">这种方法可以帮助有效地纳入各种突变的背景线粒体标记，ACP1 - GFP。它依赖于使用的机器人系统，并且可以很容易地通过使用任何机器人系统。此过程也可用于包含其他类型的标记。例如，可视化ER，我们经常使用的标记Erg11 - GFP。在我国有代表性的图片，一个突变体和野生型与ACP1 - GFP的制造商visualiz海关共聚焦显微镜研究线粒体的形态。扰乱线粒体突变体的表型，因此是一个很好的候选人作进?...

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a high-throughput method to globally study the organelle morphology in s. cerevisiae

<p class="jove_content">高通量的方法研究蛋白质的本地化或细胞器的形态，是研究蛋白质相互作用的有效工具，并能够帮助实现的分子途径全面了解。在酿酒酵母中，与非必需的基因缺失阵列发展，我们可以在全球范围研究的形态不同的细胞器如内质网（ER）和线粒体使用GFP（或变种）的标记在不同的基因背景。然而，在每一个单一的突变体含有绿色荧光蛋白标记单独是一个劳动力密集的过程。在这里，我们描述了，经常是在我们的实验室中使用的程序。我们可以通过使用一个机器人系统来处理的高密度的酵母阵列和药物筛选技术，大大缩短所需的时间，提高可重复性。总之，我们穿过一个GFP标记的线粒体标记（Apc1 GFP）的机器人副本寄托到4672不必要的基因缺失突变体的高密度阵列。通过二倍体选择，孢子形成，萌发和双标记选择，我们收回这两个等位基因。因此，每个单倍体单突变包含Apc1 - GFP在其基因组位点中。现在，我们可以研究线粒体的形态在所有非必要的突变背景。使用这种高通量的方法，我们可以方便地研究和划定的途径，并在继承和细胞器的形成，在全基因组的设置有关的基因。</p>

Watch this Scientific Journal Video about A high-throughput method to globally study the organelle morphology in S. cerevisiae at JoVE.com

A high-throughput method to globally study the organelle morphology in S. cerevisiae

<p class="jove_content">GFP融合蛋白被广泛应用于共聚焦显微镜可视化的细胞器。然而，筛选突变，影响细胞器的形态一般需要个人的突变，非常耗时。在这里，我们展示了一种方法，同时结合近5000个非必要基因在酵母细胞器- GFP标记。</p>

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Research

JoVE Journal

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科研

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Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

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