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 erkjenner støtte av Chemical Sciences, Geofag og biovitenskap Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy (pris nummer DE-FG02-91ER20021) og National Science Foundation (MCB 0.948.584) (FB). Vi er takknemlige for Ms Karen Bird for redigering av manuskriptet.

1. EMS behandling Arabidopsis thaliana frø mutagenized bruker som mutagen agenten etyl metan sulfonat (EMS) 3,4, som induserer inn i genomet C-til-T-endringer som resulterer i C / G til T / A mutasjoner 5-7. <ol><li> Vei 0,8 g Arabidopsis frø (~~~HEAD=NNS 40.000 frø) frakter organeller fluoriserende fargestoff (spesielt i denne studien ssGFPHDEL (signal sekvens-GFP-HDEL Tetrapeptid) har blitt brukt som en ER markør). </li><li> Overfør frøene i en 50-ml ...

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Her har vi beskrevet en konfokalmikroskopi-basert screening for identifisering av endomembransystemet mutanter. Denne tilnærmingen kan lett utvides til andre organeller i cellen for hvilke spesifikke fluorescerende protein markører er tilgjengelig. Skjermen er basert på identifisering av mutanter som viser en avvikende fordeling av fluoriserende fargestoff enten i mål organeller eller organeller som ikke ment å inneholde markør. Henholdsvis disse mutantene representerer bestander som enten evne organeller til subc...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Ethylmethane sulfonate </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 basal medium w Gamborg vitaminer </td><td> Phyto teknologier laboratorie </td><td> M404 </td></tr><tr><td> Phytagel </td><td> Sigma </td><td> P8169-1Kg </td></tr><tr><td> RNeasy mini kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Master ren plante blad DNA rensing kit </td><td> Epicentre </td><td> MPP92100 </td></tr><tr><td> Bioprime DNA merkesystem </td><td> Invitrogen </td><td> 18094-011 </td></tr><tr><td> Alkohol 200 proof </td><td> Decan Laboratories Inc. </td><td> 2716 </td></tr><tr><td> NaOAc </td><td> JT Baker </td><td></td></tr><tr><td> Gene chip Arabidopsis ATH1genom matrise </td><td> Affymetrix </td><td> 900385 </td></tr><tr><td> Falcon rør 50 ml </td><td> Corning </td><td> 430290 </td></tr><tr><td> Eppendorf rør 1,5 mL </td><td></td><td></td></tr><tr><td> Filter papir 90mm </td><td> Whatman </td><td> 1001090 </td></tr><tr><td> Analytisk Balanse </td><td> Mettler Toledo AB54-S </td><td> na </td></tr><tr><td> Nutating (bølge) shaker </td><td> Heidolph polymax 1040 </td><td> na </td></tr><tr><td> Sentrifuger </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

Denne protokollen beskriver en fluorescens mikroskop-basert screening av Arabidopsis frøplanter og beskriver hvordan du tilordner recessive mutasjoner som endrer subcellulære fordelingen av et bestemt merket fluoriserende fargestoff i den sekretoriske vei. Arabidopsis er en kraftig biologisk modell for genetiske studier på grunn av dens genom størrelse, generasjonstid, og bevaring av molekylære mekanismer blant kongeriker. Matrisen genotyping som en tilnærming til å kartlegge mutasjon i alternativ til den tradisjonelle metoden basert på molekylære markører er en fordel fordi det er relativt raskere og kan tillate kartlegging av flere mutanter i en veldig kort tidsramme. Denne metoden gjør at identifisering av proteiner som kan påvirke integriteten til noen organeller i planter. Her, som et eksempel, foreslår vi en skjerm å kartlegge gener som er viktige for integriteten til den endoplasmatiske retikulum (ER). Vår tilnærming, derimot, kan lett utvides til andre anlegg celleorganeller(For eksempel se 1,2), og dermed representerer et viktig skritt mot å forstå den molekylære basisen for annen subcellulære 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 grunnleggende søken i cellebiologi er å definere hvilke mekanismer som ligger til grunn for identiteten til de organeller som gjør eukaryote celler. Her foreslår vi en metode for å identifisere gener som er ansvarlige for den morfologiske og funksjonelle integritet av plante organeller med fluorescens mikroskopi og neste generasjons sekvensering verktøy.

Fluorescens-mikroskopi Screening og neste generasjons sekvensering: Nyttige verktøy for identifikasjon av gener involvert i Organelle 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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