高通量酵母质粒过表达屏幕
Summary
在这里，我们描述了一个质粒的过度表达屏幕<em>酿酒酵母</em>，使用液体处理机器人摆着质粒库和高通量酵母转化协议。
Abstract
芽殖酵母， 酿酒酵母 ，是一个强大的模型系统，用于定义许多重要的细胞过程，包括那些与人类疾病直接相关的基本机制。由于其短的一代人的时间和特点是基因组，实验酵母模型系统的一个主要优势是能够进行遗传筛选，以确定基因和途径，是在一个给定的过程中涉及的。在过去的三十多年，这种遗传屏幕已被用来阐明细胞周期，分泌途径，以及更高度保守的许多方面的真核细胞生物学1-5。在过去的几年里中，几个酵母菌株和质粒基因组库已产生 6-10 。这些藏品现在允许对基因功能的系统，使用增益和损失功能的方法11-16的审讯。在这里，我们提供了一个详细的协议，与液体处理机器人执行质粒的过度表达屏幕使用一种高通量的酵母转化协议使用5500酵母质粒摆着库。我们一直在使用这些画面，找出容易发生聚集人类神经退行性疾病的蛋白质的积累相关的遗传毒性修饰符。这里介绍的方法很容易适应其他感兴趣的细胞表型的研究。
Protocol
1。酵母转化的筹备工作此协议是专为10个96孔板，但可以向上或向下扩展。我们发现，这个协议不超过二96孔盘每一轮的转型工作。整个改造过程（步骤I.3），将需要约8小时。 分装到每一个圆底96孔板Biorobot RapidPlate液体处理程序以及FLEXGene从酵母的ORF库的质粒DNA（为100 ng /μL）5 -10μL。放置在洁净工作台，通风柜一夜之间干破获96孔板。我们已经发现类似与CEN和2μ酵母?…
Discussion
在这里，我们提出了一个协议来执行一种高通量质粒在酵母中的过度表达屏幕。这种方法可以让许多不同的细胞表型的遗传修饰词的迅速和公正的筛选。使用这种方法，研究人员可以在几个星期内屏幕的酵母基因组中的一个重要部分。这种不偏不倚的方式也可以修饰词的识别，预测基于以前的研究结果可能没有被。我们已经用这种方法来确定与人类神经退行性疾病的蛋白质的聚集 19,21-23?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由惠普ALS研究中心的约翰霍普金斯（ADG），美国国立卫生研究院主任的新的创新奖1DP2OD004417 – 01（ADG），美国国立卫生研究院R01 NS065317（ADG），丽塔艾伦基金会学者奖授予的支持。助理总干事，是由皮尤慈善信托基金支持的“皮尤研究中心的学者，在生物医学科学”。
Materials
| Name of reagent | Company | Catalog number |
|---|---|---|
| BioRobot RapidPlate | Qiagen | 9000490 |
| 96 bolt replicator (frogger) | V&P Scientific | VP404 |
| FLEXGene ORF Library | Institute of Proteomics, Harvard Medical School | |
| Tabletop centrifuge | Eppendorf | 5810R |
| 500mL baffled flask | Bellco | 2543-00500 |
| 2.8L triple-baffled Fernbach flask | Bellco | 2551-02800 |
| 100μL Rapidplate pipette tips | Axygen | ZT-100-R-S |
| 200μL Rapidplate pipette tips | Axygen | ZT-200-R-S |
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Cite This Article
Fleming, M. S., Gitler, A. D. High-throughput Yeast Plasmid Overexpression Screen. J. Vis. Exp. (53), e2836, doi:10.3791/2836 (2011).