This work was supported by a grant from the Packard Center for ALS Research at Johns Hopkins (A.D.G.), an NIH Director&#x2019;s New Innovator Award 1DP2OD004417-01 (A.D.G), NIH R01 NS065317 (A.D.G.), the Rita Allen Foundation Scholar Award. A.D.G. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts.

1.	Preparations for yeast transformation 
This protocol is designed for ten 96-well plates but can be scaled up or down accordingly. We have found that this protocol does not work well for more than twenty 96-well plates per round of transformation. The entire transformation procedure (from step I.3) will take approximately eight hours.
<ol>
<li>Aliquot 5-10&mu;L of plasmid DNA (100 ng/&mu;l) from the Yeast FLEXGene ORF library into each well of a round-bottom 96-well plate with Biorobot Rap...

<ol>
	<li>Nurse, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nobel+Prize+and+beyond:+an+interview+with+Sir+Paul+Nurse.+Interview+by+Susan+R.+Owens.">The Nobel Prize and beyond: an interview with Sir Paul Nurse. Interview by Susan R. Owens.</a> EMBO Rep. 3, 204-206 (2002).</li><li>Hartwell, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nobel+Lecture.+Yeast+and+cancer.">Nobel Lecture. Yeast and cancer.</a> Biosci Rep. 22, 373-394 (2002).</li><li>Stevens, T., Esmon, B., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+in+the+yeast+secretory+pathway+are+required+for+transport+of+carboxypeptidase+Y+to+the+vacuole.">Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole.</a> Cell. 30, 439-448 (1982).</li><li>Novick, P., Ferro, S., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+of+events+in+the+yeast+secretory+pathway.">Order of events in the yeast secretory pathway.</a> Cell. 25, 461-469 (1981).</li><li>Novick, P., Field, C., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+23+complementation+groups+required+for+post-translational+events+in+the+yeast+secretory+pathway.">Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway.</a> Cell. 21, 205-215 (1980).</li><li>Sopko, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+pathways+and+phenotypes+by+systematic+gene+overexpression.">Mapping pathways and phenotypes by systematic gene overexpression.</a> Mol Cell. 21, 319-330 (2006).</li><li>Alberti, S., Gitler, A. D., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suite+of+Gateway((R))+cloning+vectors+for+high-throughput+genetic+analysis+in+Saccharomyces+cerevisiae.">A suite of Gateway((R)) cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.</a> Yeast. 24, 913-919 (2007).</li><li>Gelperin, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+genetic+analysis+of+the+yeast+proteome+with+a+movable+ORF+collection.">Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.</a> Genes Dev. 19, 2816-2826 (2005).</li><li>Hu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaching+a+complete+repository+of+sequence-verified+protein-encoding+clones+for+Saccharomyces+cerevisiae.">Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.</a> Genome Res. 17, 536-543 (2007).</li><li>Giaever, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+profiling+of+the+Saccharomyces+cerevisiae+genome.">Functional profiling of the Saccharomyces cerevisiae genome.</a> Nature. 418, 387-391 (2002).</li><li>Boone, C., Bussey, H., Andrews, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+genetic+interactions+and+networks+with+yeast.">Exploring genetic interactions and networks with yeast.</a> Nat Rev Genet. 8, 437-449 (2007).</li><li>Mnaimneh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+essential+gene+functions+via+titratable+promoter+alleles.">Exploration of essential gene functions via titratable promoter alleles.</a> Cell. 118, 31-44 (2004).</li><li>Parsons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+chemical-genetic+and+genetic+interaction+data+links+bioactive+compounds+to+cellular+target+pathways.">Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.</a> Nat Biotechnol. 22, 62-69 (2004).</li><li>Schuldiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+the+function+and+organization+of+the+yeast+early+secretory+pathway+through+an+epistatic+miniarray+profile.">Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.</a> Cell. 123, 507-519 (2005).</li><li>Tong, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+genetic+analysis+with+ordered+arrays+of+yeast+deletion+mutants.">Systematic genetic analysis with ordered arrays of yeast deletion mutants.</a> Science. 294, 2364-2368 (2001).</li><li>Tong, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+mapping+of+the+yeast+genetic+interaction+network.">Global mapping of the yeast genetic interaction network.</a> Science. 303, 808-813 (2004).</li><li>Neumann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitinated+TDP-43+in+frontotemporal+lobar+degeneration+and+amyotrophic+lateral+sclerosis.">Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.</a> Science. 314, 130-133 (2006).</li><li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. 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+yeast+TDP-43+proteinopathy+model:+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity.">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proc Natl Acad Sci U S A. 105, 6439-6444 (2008).</li><li>Elden, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ataxin-2+intermediate-length+polyglutamine+expansions+are+associated+with+increased+risk+for+ALS.">Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.</a> Nature. 466, 1069-1075 (2010).</li><li>Johnson, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+is+intrinsically+aggregation-prone,+and+amyotrophic+lateral+sclerosis-linked+mutations+accelerate+aggregation+and+increase+toxicity.">TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity.</a> J Biol Chem. 284, 20329-20339 (2009).</li><li>Cooper, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313, 324-328 (2006).</li><li>Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beer+and+Bread+to+Brains+and+Beyond:+Can+Yeast+Cells+Teach+Us+about+Neurodegenerative+Disease?.">Beer and Bread to Brains and Beyond: Can Yeast Cells Teach Us about Neurodegenerative Disease?.</a> Neurosignals. 16, 52-62 (2008).</li><li>Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+is+part+of+a+diverse+and+highly+conserved+interaction+network+that+includes+PARK9+and+manganese+toxicity.">Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity.</a> Nat Genet. 41, 308-315 (2009).</li></ol>

No conflicts of interest declared.

Here we present a protocol to perform a high-throughput plasmid overexpression screen in yeast. This approach allows for the rapid and unbiased screening for genetic modifiers of many different cellular phenotypes. Using this approach, a researcher can screen a significant portion of the yeast genome in a matter of weeks. This unbiased approach also allows for the identification of modifiers, which may not have been predicted based on previous findings. We have used this approach to identify modifiers of toxicity assoc...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of reagent</th>
 <th>Company</th>
 <th>Catalog number</th>
 </tr>
 <tr>
 	<td>BioRobot RapidPlate</td>
 <td>Qiagen</td>
 <td>9000490</td>
 </tr>
 <tr>
 	<td>96 bolt replicator (frogger)</td>
 <td>V&amp;P Scientific</td>
 <td>VP404</td>
 </tr>
 <tr>
 	<td>FLEXGene ORF Library</td>
 <td>Institute of Proteomics, Harvard Medical School</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Tabletop centrifuge</td>
 <td>Eppendorf</td>
 <td>5810R</td>
 </tr>
 <tr>
 	<td>500mL baffled flask</td>
 <td>Bellco</td>
 <td>2543-00500</td>
 </tr>
 <tr>
 	<td>2.8L triple-baffled Fernbach flask</td>
 <td>Bellco</td>
 <td>2551-02800</td>
 </tr>
 <tr>
 	<td>100&mu;L Rapidplate pipette tips</td>
 <td>Axygen</td>
 <td>ZT-100-R-S</td>
 </tr>
 <tr>
 	<td>200&mu;L Rapidplate pipette tips</td>
 <td>Axygen</td>
 <td>ZT-200-R-S</td>
 </tr>
 </tbody>
</table>

high-throughput yeast plasmid overexpression screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

high-throughput-yeast-plasmid-overexpression-screen

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JoVE Journal

Biology

16.3K ビュー.  University of Pennsylvania School of Medicine. ここではでプラスミド過剰発現の画面を説明サッカロマイセスセレビシエ、アレイプラスミドライブラリーと液体ハンドリングロボットによるハイスループット酵母の形質転換プロトコルを使用して。