Name: fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity
Uploaded: 2012-04-13T12:00:00
Description: Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Vi erkänner stöd från Chemical Sciences, geovetenskap och Biosciences Division Office of Basic Energy Sciences, Office of Science, US Department of Energy (tilldelning antalet DE-FG02-91ER20021) och National Science Foundation (MCB 0.948.584) (FB). Vi är tacksamma för att Karen Bird för redigering av manuskriptet.

1. EMS Behandling Arabidopsis thaliana frön mutageniseras med användning som mutagent medel etylmetansulfonat (EMS) 3,4, vilket inducerar i genomet C-till T-förändringar som resulterar i C / G till T / A mutationer 5-7. <ol><li> Väg 0,8 g Arabidopsis frön (cirka 40.000 frön) som bär organellen fluorescerande markör (bestämt i denna studie ssGFPHDEL (signalsekvens-GFP-HDEL tetrapeptid) har använts som en ER-markör). </li><li> Överföra fröna i en...

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Här har vi beskrivit en konfokal mikroskopi screening för identifiering av endomembrane mutanter. Detta tillvägagångssätt kan enkelt utvidgas till andra organeller i cellen för vilken särskilda fluorescerande proteinmarkörer finns tillgängliga. Skärmen är baserad på identifieringen av mutanter som visar en avvikande fördelning av den fluorescerande markören antingen i mål-organell eller organeller som inte är tänkta att innehålla markören. Respektive dessa mutanter representerar populationer då antin...

<table border="1"><tbody><tr><td> Namnet på reagens </td><td> Företaget </td><td> Katalognummer </td></tr><tr><td> Etylmetansulfonat </td><td> Sigma </td><td> M0880 </td></tr><tr><td> NaOH </td><td> JT Baker </td><td> 3722-05 </td></tr><tr><td> Murashige Skoog basalmedium w Gamborg vitaminer </td><td> Phyto Technolog laboratorie </td><td> M404 </td></tr><tr><td> Phytagel </td><td> Sigma </td><td> P8169-1 kg </td></tr><tr><td> RNeasy Mini Kit </td><td> Qiagen </td><td> 74.104 </td></tr><tr><td> Mästare ren vegetabilisk blad DNA reningskit </td><td> Epicentrum </td><td> MPP92100 </td></tr><tr><td> Bioprime DNA märkningssystem </td><td> Invitrogen </td><td> 18094-011 </td></tr><tr><td> Alkohol 200 proof </td><td> Dekan laboratorier inkl. </td><td> 2716 </td></tr><tr><td> NaOAc </td><td> JT Baker </td><td></td></tr><tr><td> Genchip Arabidopsis ATH1genomet matris </td><td> Affymetrix </td><td> 900.385 </td></tr><tr><td> Falcon-rör 50 ml </td><td> Corning </td><td> 430.290 </td></tr><tr><td> Eppendorf-rör 1,5 ml </td><td></td><td></td></tr><tr><td> Filterpapper 90mm </td><td> Whatman </td><td> 1001090 </td></tr><tr><td> Analysvåg </td><td> Mettler Toledo AB54-S </td><td> na </td></tr><tr><td> Nuterande (våg) shaker </td><td> Heidolph Polymax 1040 </td><td> na </td></tr><tr><td> Centrifugera </td><td> Eppendorf 5417-R </td><td> na </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

Detta protokoll beskriver en fluorescens-mikroskop-baserad screening av Arabidopsis plantor och beskriver hur man mappa recessiva mutationer som förändrar den subcellulära fördelningen av en märkt specifik fluorescerande markör i den sekretoriska vägen. Arabidopsis är en kraftfull biologisk modell för genetiska studier på grund av dess genom storlek, generationstid, och bevarande av molekylära mekanismer bland riken. Arrayen genotypning som en metod att kartlägga mutationen i alternativ till den traditionella metod som bygger på molekylära markörer är fördelaktigt eftersom det är relativt snabbare och kan tillåta kartläggning av flera mutanter i en riktigt kort tid. Denna metod gör det möjligt att identifiera proteiner som kan påverka integriteten hos all organell i växter. Här, som ett exempel, föreslår vi en skärm för att kartlägga gener som är viktiga för integriteten hos det endoplasmatiska retiklet (ER). Vårt tillvägagångssätt, kan dock lätt utvidgas till andra organeller växt cell(Se till exempel 1,2), och utgör därmed ett viktigt steg mot att förstå den molekylära grunden för övriga subcellulära strukturer.

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

En grundläggande strävan i cellbiologi är att definiera de mekanismer som ligger bakom identiteten av organeller som gör eukaryota celler. Här föreslår vi en metod för att identifiera de gener som är ansvariga för den morfologiska och funktionella integritet växt organeller med fluorescensmikroskopi och nästa generation verktyg sekvensering.

Fluorescens-mikroskopi Screening och nästa generations sekvensering: Användbara verktyg för identifiering av gener involverade i Organell Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

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