Name: flow cytometry-based purification of s. cerevisiae zygotes
Uploaded: 2012-09-21T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry-based Purification of S. cerevisiae Zygotes at JoVE.com

我们感谢R.迈克尔Sramkoski博士的帮助，流式细胞仪，主任Purnima Jai​​swal博士和博士翟武以前的输入和伊利亚·Aylyarov插图的帮助。支持的由NIH资助R01GM089872的和流式细胞术和显微成像技术的核心设施的案例综合癌症中心（P30 CA43703）。

1。单倍体细胞生长<ol><li>成长S.从的S288C背景（BY4741）的酵母细胞在标准条件下10。 </li><li>变换单倍体两个显眼的荧光标记物10的任一表达。给出的标记物是在细胞质中表达的可溶性的GFP（pEG220，URA3/YIp，11用BglII线性化）和一个单一的核糖体蛋白，RPL25 mCherry（pAJ1661，URA3/CEN，从A约翰斯顿）融合。我们预计，相当于两色的纯化方法可以利用其他明亮的荧光蛋?...

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	<li>Herskowitz, I., Oshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Control of cell type in Saccharomyces cerevisiae: Mating type and mating-type interconversion. , 181-210 (1981).</li><li>Muller, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parental+age+and+the+life-span+of+zygotes+of+Saccharomyces+cerevisiae.">Parental age and the life-span of zygotes of Saccharomyces cerevisiae.</a> Antonie Van Leeuwenhoek. 51, 1-10 (1985).</li><li>Tartakoff, A. M., Jaiswal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+fusion+and+genome+encounter+during+yeast+zygote+formation.">Nuclear fusion and genome encounter during yeast zygote formation.</a> Mol. Biol. Cell. 20, 2932-2942 (2009).</li><li>Azpiroz, R., Butow, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+mitochondrial+sorting+in+yeast+zygotes.">Patterns of mitochondrial sorting in yeast zygotes.</a> Mol. Biol. Cell. 4, 21-36 (1993).</li><li>Rose, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+fusion+in+the+yeast+Saccharomyces+cerevisiae.">Nuclear fusion in the yeast Saccharomyces cerevisiae.</a> Annu. Rev. Cell Dev. Biol. 12, 663-695 (1996).</li><li>Dujon, B., Jones, E., Strathern, J., Broach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. , 505-635 (1981).</li><li>Sprague, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+yeast+mating+reaction.">Assay of yeast mating reaction.</a> Methods Enzymol. 194, 77-93 (1991).</li><li>Ydenberg, C. A., Rose, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mating:+a+model+system+for+studying+cell+and+nuclear+fusion.">Yeast mating: a model system for studying cell and nuclear fusion.</a> Methods Mol. Biol. 475, 3-20 (2008).</li><li>Sena, E. P., Radin, D. N., Fogel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+mating+in+yeast.">Synchronous mating in yeast.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 1373-1377 (1973).</li><li>Amberg, D., Burke, D., Strathern, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2005 Edition. , (2005).</li><li>Nolan, S., Cowan, A. E., Koppel, D. E., Jin, H., Grote, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FUS1+regulates+the+opening+and+expansion+of+fusion+pores+between+mating+yeast.">FUS1 regulates the opening and expansion of fusion pores between mating yeast.</a> Mol. Biol. Cell. 17, 2439-2450 (2006).</li><li>Weisman, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelles+on+the+move:+insights+from+yeast+vacuole+inheritance.">Organelles on the move: insights from yeast vacuole inheritance.</a> Nat. Rev. Mol. Cell Biol. 7, 243-252 (2006).</li></ol>

获得纯化的受精卵应包括转录组学，蛋白质组学和脂类组学，以及调查机制，促进生化研究的受精卵进行重新进入细胞周期，细胞壁重塑，萌芽和继承的特点，另外，受精卵可以开始生成与承载特性突变株在父母一方或双方。此外，纯化的受精卵，为单倍体或二倍体细胞，也被冻结在含15％甘油的培养基和购买解冻监察方面经常出芽。 使用其他适合微观目的的荧光标记物（ <e...

<table border="1"><tbody><tr><td> 试剂/设备 </td><td> 源 </td><td> 目录编号 </td></tr><tr><td> pEG220，URA3/YIp，编码细胞质GFP 11） </td><td> E.格罗特</td><td></td></tr><tr><td> pAJ1661，URA3/CEN，RPL25编码融合mCherry </td><td> A.约翰斯顿</td><td></td></tr><tr><td>费舍尔FB120超声波仪</td><td> Fisher Scientific则</td><td></td></tr><tr><td>聚丙烯管滤网盖</td><td> BD猎鹰</td><td>参考352235 </td></tr><tr><td>冷藏的离心</td><td> TOMY </td><td> MRX-150 </td></tr><tr><td>流式细胞仪鞘液</td><td> Biosure </td><td> 1027 </td></tr><tr><td> iCyt反射模型流式细胞仪</td><td>索尼</td><td></td></tr><tr><td> DeltaVision的反卷积显微镜</td><td>磷肥recision </td><td></td></tr><tr><td>直立式落射荧光显微镜</td><td>徕卡</td><td></td></tr><tr><td>超纯琼脂糖</td><td> Invitrogen公司</td><td> 15510-07 </td></tr><tr><td> CSM葡萄糖配方 注：如果在琼脂平板上，简单地包括2％琼脂（Difco） </td><td> 10 </td><td></td></tr></tbody></table>

flow cytometry-based purification of s. cerevisiae zygotes

合子是必不可少的中间体单倍体和二倍体国家之间在许多生物的生命周期，包括酵母（ 图1）1。S. 酵母合子从不同的交配型的单倍体细胞（MATa的，MATalpha）的融合的结果，并产生相应的稳定的二倍体，依次产生多达20二倍体后代作为结果惊人地不对称的有丝分裂2。 Zygote的形成是由一个复杂的事件序列策划：在这个过程中，可溶性交配因子结合到同源受体，触发受体介导的信号转导通路的有利于在细胞 - 细胞融合的细胞周期和达到高潮中断。受精卵可考虑的祖代细胞或干细胞的功能的模型。 虽然已经了解到的形成受精卵，受精卵已用于研究细胞分子的问题，一般显“接力，几乎所有的研究都使用的交配混合物，其中的受精卵混合与人口占多数的单倍体细胞3-8。合子形成的生物化学的研究的许多方面和合子继续寿命，因此，仍然得不到澄清。 报告的纯化酵母受精卵的描述协议根据其沉降性，但是，这种沉淀为基础的程序没有取得近90％的纯度在我们手中。此外，它具有的缺点，即细胞暴露于高渗山梨糖醇。因此，我们已经开发出一种通用的纯化方法。为了这个目的，双单倍体细胞表达红色或绿色荧光蛋白共同孵育，以允许合子形成，在不同时间收获的，和所得到的受精卵进行纯化，使用流式细胞仪基于排序协议。这种技术提供了一个方便的可视化评估的纯度和成熟。平均纯度的馏分，是约90％。根据收获的时间，受精卵的成熟程度不一，可以恢复。纯化的样品提供了一个方便的“组学”研究的出发点，为恢复初始后代，这个祖细胞，并进行系统的调查。

Watch this Scientific Journal Video about Flow Cytometry-based Purification of S. cerevisiae Zygotes at JoVE.com

Flow Cytometry-based Purification of S. cerevisiae Zygotes

为了净化受精卵<em&gt; S.酵母</em&gt;，相反交配型的单倍体细胞的设计，以表达红色或绿色荧光蛋白，共同孵育，以允许合子形成，并分级的使用基于流式细胞术的协议。高度富集的部分，随后的“组学”的研究，恢复初始的后代，受精卵形态和系统的调查。

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

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

Research

JoVE Journal

Biology

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Assay for Adhesion and Agar Invasion in S. cerevisiae

化验粘附和酿酒酵母琼脂入侵

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

科研

生物学

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

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 ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Flow Cytometry Purification of Mouse Meiotic Cells

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

High Throughput Microinjections of Sea Urchin Zygotes

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...