Name: high-throughput yeast plasmid overexpression screen
Uploaded: 2011-07-27T13:00:00
Description: Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

यह काम जॉन्स हॉपकिन्स पर पैकार्ड ए एल एस अनुसंधान के लिए केंद्र (एडीजी), एक NIH निदेशक का नया अन्वेषक पुरस्कार 1DP2OD004417 01 - (एडीजी), एनआईएच R01 NS065317 (एडीजी), रीता एलन फाउंडेशन विद्वान पुरस्कार से एक अनुदान द्वारा समर्थित किया गया था. एडीजी बायोमेडिकल साइंसेज में एक बेंच विद्वान, बेंच चैरिटेबल ट्रस्ट द्वारा समर्थित है.

1. खमीर परिवर्तन के लिए तैयारी इस प्रोटोकॉल दस 96 अच्छी तरह से प्लेटों के लिए डिज़ाइन किया गया है, लेकिन ऊपर या नीचे बढ़ाया जा सकता है है तदनुसार. हमने पाया है कि इस प्रोटोकॉल अच्छी तरह से बीस से अ?...

<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>

यहाँ हम एक प्रोटोकॉल करने के लिए खमीर में एक उच्च throughput प्लाज्मिड overexpression स्क्रीन प्रदर्शन मौजूद है. इस दृष्टिकोण तेजी से और निष्पक्ष जांच के लिए कई अलग अलग सेलुलर phenotypes की आनुवंशिक संशोधक के लिए अनुमति देत?...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचि संख्या </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 बोल्ट replicator (Frogger) </td><td> वी पी एंड वैज्ञानिक </td><td> VP404 </td></tr><tr><td> FLEXGene ओआरएफ पुस्तकालय </td><td> प्रोटिओमिक्स संस्थान, हार्वर्ड मेडिकल स्कूल </td><td></td></tr><tr><td> Tabletop अपकेंद्रित्र </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500ml चकित फ्लास्क </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L ट्रिपल चकित Fernbach फ्लास्क </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μL Rapidplate विंदुक युक्तियाँ </td><td> Axygen </td><td> ZT 100 रु </td></tr><tr><td> 200μL Rapidplate विंदुक युक्तियाँ </td><td> Axygen </td><td> ZT 200 रु </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

नवोदित खमीर Saccharomyces cerevisiae, मानव रोग के लिए प्रत्यक्ष प्रासंगिकता के साथ उन सहित कई महत्वपूर्ण सेलुलर प्रक्रियाओं के बुनियादी तंत्र को परिभाषित करने के लिए एक शक्तिशाली मॉडल प्रणाली है. अपनी छोटी पीढ़ी समय और अच्छी तरह से विशेषता जीनोम की वजह से, खमीर मॉडल प्रणाली के एक प्रमुख प्रयोगात्मक लाभ के लिए आनुवंशिक स्क्रीन प्रदर्शन करने के लिए जीन और मार्ग है कि किसी भी प्रक्रिया में शामिल हैं की पहचान करने की क्षमता है. पिछले तीस साल में इस तरह के आनुवंशिक स्क्रीन सेल चक्र, स्रावी मार्ग, और यूकेरियोटिक सेल 1-5 जीव विज्ञान के कई अधिक उच्च संरक्षित पहलुओं को स्पष्ट करने के लिए इस्तेमाल किया गया है. पिछले कुछ वर्षों में, खमीर उपभेदों और plasmids के कई genomewide पुस्तकालयों 6-10 उत्पन्न किया गया है. इन संग्रहों में अब जीन समारोह के व्यवस्थित लाभ और हानि के समारोह 11-16 दृष्टिकोण का उपयोग कर पूछताछ के लिए अनुमति देते हैं . यहाँ हम एक तरल से निपटने रोबोट एक प्लाज्मिड overexpression स्क्रीन प्रदर्शन के साथ उच्च throughput एक खमीर परिवर्तन प्रोटोकॉल के उपयोग के लिए विस्तृत प्रोटोकॉल प्रदान करते हैं, 5500 खमीर plasmids के एक arrayed पुस्तकालय का उपयोग कर. हम इन स्क्रीन का उपयोग किया गया एकत्रीकरण प्रवण मानव neurodegenerative रोग प्रोटीन के संचय के साथ जुड़े विषाक्तता के आनुवंशिक संशोधक की पहचान. यहाँ प्रस्तुत तरीकों को आसानी से ब्याज की अन्य सेलुलर phenotypes के अध्ययन के लिए अनुकूलनीय रहे हैं.

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

High-throughput Yeast Plasmid Overexpression Screen

यहाँ हम में एक प्लाज्मिड overexpression स्क्रीन का वर्णन<em&gt; Saccharomyces cerevisiae</em&gt; एक तरल से निपटने रोबोट के साथ एक arrayed प्लाज्मिड पुस्तकालय और उच्च throughput एक खमीर परिवर्तन प्रोटोकॉल का उपयोग.

उच्च throughput खमीर प्लाज्मिड Overexpression स्क्रीन

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

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