Name: an efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in arabidopsis
Uploaded: 2014-06-19T14:00:00
Description: Watch this Scientific Journal Video about An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis at JoVE.com

We thank Ueli Grossniklaus (University of Z&#252;rich) for technical and financial support. We are thankful to Valeria Gagliardini, Christof Eichenberger, Arturo Bolanos and Peter Kopf for general lab support. This research was funded by the University of Z&#252;rich, grants from the Swiss National Foundation to CB (31003A_130722) and Ueli Grossniklaus (31003A_141245 and 31003AB-126006), and the Agence Nationale de la Recherche to DG (Programme ANR-BLANC-2012).

Proceduren er beskrevet i arbejdsgangen i figur 1, og opsætningen for dissektion og indlejring af væv er vist i figur 2.. 1. Vævsfiksering <ol><li> Saml 20-30 Frugtbladene i et mikrocentrifugerør med frisk gjort BVO fiksativ buffer på is. </li><li> Lave vævet 30 min med forsigtig rystning ved stuetemperatur. </li><li> Spin rørene indeholdende frugtblade i fiksativ i en benchtop mikrocentrifuge 1 min ved 400 x g. </li><li> Fjern omhyggeligt Fikservæ...

<ol>
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I blomstrende planter, er den kvindelige reproduktive afstamning omgivet af flere cellelag, herunder nucellus og ovule teguments, hvilket gør cytologisk farvning i hel-mount teknisk udfordrende. Her præsenterer vi en effektiv protokol muliggør fremstilling og forarbejdning af et stort antal af frøanlæg egnet til cytologisk farvning såsom immunfarvning, DNA-farvning og fluorescens in situ hybridisering i hel-mount. Vi har med held brugt det til analyse af den kvindelige reproduktive germline i Arabidop...

I blomstrende planter, etablering af reproduktive slægter begynder med differentieringen af ​​SMC&#39;er, kvindelige MMC og mandlige mikrosporeudvikling mor celle. MMC udvikler sig fra en sub-epidermal frøkrop celle ved den distale spids af ovule primordium og mikrosporeudvikling mor cellen udvikler sig fra sporedannende væv i støvknappen locule, som er placeret dybt inde i de blomsteragtige organer 1. SMC&#39;er undergår meiose at producere haploide sporer, som så giver anledning til gametophytes upon mitose. Den kvindelige gametofyt eller foster sæk, består af en ægcelle, en central celle, to synergids og tre antipodals. Den mandlige gametofyt eller pollen, der består af en vegetativ celle og to sædceller. Mens den mandlige gametofyt fortsat en relativt tilgængelig objekt-of-undersøgelse, er den kvindelige gametofyt indlejret i ovule, selv indesluttet i blomsten frugtblad, og dermed giver særlige udfordringer til molekylære og cytologiske analyser. For nylig, men laser-assisteretmikrodissektion tilbudt en elegant løsning, der tillader transkriptom analyser i MMC og kvindelige gametophytic celler 2-4. Ud over kandidat genekspression analyser, ved hjælp af fx RNA in situ hybridisering eller reportergenassays, cytologiske analyser tillader at undersøge dynamikken i endogene cellulære komponenter ved hjælp af specifik direkte cellulær farvning eller indirekte immunfarvning. Især cytogenetisk farvning bruge FISH og DNA-farvning, sammen med immunfarvning af kromatin ændringer eller kromatin komponenter er centrale tilgange at belyse kromatin dynamik og nukleare organisation i Arabidopsis 5.. Typisk meiose indebærer særlige kromosomfejl dynamik, der er blevet godt undersøgt i plante mandlige meiocytter 6,7; yderligere storstilede, celle-specifikke kromatin reorganisering, sandsynligvis afspejler dynamisk epigenetiske omprogrammering er blevet beskrevet under pollen udvikling 8-10. Derimod grundtil den relative utilgængelighed af den kvindelige meiocyte og gametofyt forbliver disse undersøgelser teknisk vanskeligt at anvende, og kræver ofte sektionering eller manuel dissektion og enzymatisk nedbrydning (se nedenfor). Hertil kommer, at fremherskende mangel på optisk klarhed i hel-mount er en hindring for høj opløsning billeddannelse af kønsceller i intakte frøanlæg. En klassisk metode til cytologisk analyse af kromosom organisation i hele-mount frøanlæg bruger Feulgen s farvning 11-13. Det indebærer syrehydrolyse (ved hjælp af hypochlorsyrling) af DNA, hvilket resulterer i proteindenaturering og således forårsager ødelæggelse af kromatin. Alternativt kan kromosom organisation i kvindelige meiocytter og gametophytic celler observeret ved hjælp DAPI farvning og immunfarvning på semi-tynde sektioner eller dissekeret embryo sække og MMC (se for eksempel 14-18). Imidlertid klart, at manuel dissektion og skæring kan være arbejdskrævende oghæmmer den kvalitative og kvantitative analyse af et stort antal af kromatin epitoper. Her giver vi en effektiv protokol til at forberede et stort antal Arabidopsis frøanlæg egnet til en række aftagere cytologisk farvning i hel-mount. Kort sagt, er blomsterknopper inkuberes i en fikseringsopløsning, der rækker af frøanlæg dissekeret fra frugtbladet og indlejret i acrylamid på dias som gjort for pollen meiocytter 19,20. Den indlejrede frøanlæg yderligere ryddet og fikseret i methanol, ethanol og xylen før cellevæg fordøjelse og permeabilisering. Mulige variationer af disse trin diskuteres. Prøverne kan derefter anvendes til DNA-farvning, immunfarvning og FISH. Præparatet tilstand er effektiv og giver mulighed for parallel forsøgsopstilling (op til 16 slides kan være forberedt på en dag for forskellige nedstrøms analyse). De beskrevne behandlinger muliggøre homogene signaler i hel-mount og velbevarede histologiske, cellulære,og nuklear organisation i reproduktive celler og omkringliggende frøkrop celler, der gavner kvalitative og kvantitative sammenligninger mellem celletyper. Kalibreret, CLSM-baserede høj opløsning billeddannelse efterfulgt af 3-dimensionel rekonstruktion muliggør meningsfyldte kvantitative målinger af fluorescerende signaler. Vi har med held brugt denne fremgangsmåde til at analysere kromatin dynamik i den differentierende MMC 21 og udvikle kvindelige gametofyt 22; vi præsenterer her repræsentative resultater af heterochromatin analyse, kromatin immunfarvning, GFP immunfarvning og FISH i hel-mount frøanlæg. Vi mener endvidere, at vores protokol vil være egnet til andre plantevæv og arter.

<table border="0" cellpadding="0" cellspacing="0" width="993">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"><a name="RANGE!A1:D39">Name of Material/ Equipment</a></td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:481px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Solutions</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BVO Fixation Buffer (based on32)</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">2mM EGTA, pH7.5, 1% (v/v) Formaldehyde,10% DMSO, 1&#215; PBS, 0.1% Tween-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBT</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">1&#215; PBS, 0.1% Tween-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBT-F</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">1&#215; PBT, 2.5% (v/v) formaldehyde</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">30% acrylamide:bisacrylamide&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">3g acrylamide, 0.33g bisacrylamide, 1&#215; PBS (prepare 10ml, store at 4&#176;C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">200mL&#160; 5% acrylamide mix in PBS&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">34mL 30% acrylamide:bisacrylamide, 166mL 1&#215; PBS (make fresh from 30% stock)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">20% ammoniumpersulfte&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.2g ammoniumpersulfte, 1mL sterile water (prepare aliquots with 1mL and store at -20&#176;C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">20% sodium sulfite&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.2g sodium sulfite, 1mL sterile water (prepare aliquots with 1mL and store at -20&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell wall enzyme mix</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.5% (w/v) cellulase, 1% (w/v) driselase, 0.5% (w/v) pectolyase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents and Materials</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F1635</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Amaresco</td>
			<td>0497</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Schaurlau</td>
			<td>ET00102500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Schaurlau</td>
			<td>ME03062500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>ROTH</td>
			<td>4436.1</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase</td>
			<td>Sigma</td>
			<td>1794</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Driselase</td>
			<td>Sigma</td>
			<td>D8037</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pectolyase</td>
			<td>Sigma</td>
			<td>P5936</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Merck</td>
			<td>8.22184.0500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td>Sigma</td>
			<td>E-4378</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acrylamide</td>
			<td>Sigma</td>
			<td>A-3553</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bisacrylamide</td>
			<td>Sigma</td>
			<td>M2022</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammoniumpersulfate</td>
			<td>Sigma</td>
			<td>A9164</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium sulfite</td>
			<td>Fluka</td>
			<td>71988</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-trimethyl-Histone H3 (Lys4)</td>
			<td>Upstate</td>
			<td>07-473</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti- monomethyl-Histone H3 (Lys27)</td>
			<td>Upstate</td>
			<td>07-448</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488~goat ~anti ~rabbit (H+L)</td>
			<td>Molecular Probe</td>
			<td>A11008</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProlongGold</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse A</td>
			<td>Roche</td>
			<td>10109169001</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coplin jar</td>
			<td>Huber &#38; CO</td>
			<td>10.055</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>DUMONT BIOLOGY</td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker</td>
			<td>Heidolph</td>
			<td>543-12310-00-0</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Moist chamber</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">A plastic box with damp paper towel inside, a plastic support is put into the box for supporting the slides and keep slides from the water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus slide</td>
			<td>Thermo Fisher</td>
			<td>J1800AMNZ</td>
			<td style="width:481px;">Menzel-Gl&#228;ser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FISH Tag DNA KIt</td>
			<td>Invitrogen</td>
			<td>F32947</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GFP booster</td>
			<td style="width:135px;">Chromotek</td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
	</tbody>
</table>

an efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in arabidopsis

I blomstrende planter, er det somatiske-til-reproduktive celle skæbne overgang præget af specifikationen af ​​spore mor celler (SMC&#39;er) i blomstermotiver organer i voksen plante. Den kvindelige SMC (megaspore mor celle, MMC) skelner i ovule primordium og undergår meiose. Den valgte haploide megaspore gennemgår derefter mitose at danne flercellede kvindelige gametofyt, hvilket vil give anledning til kønsceller, ægcellen og centrale celle sammen med accessoriske celler. Den begrænsede tilgængelighed af MMC, meiocyte og kvindelige gametofyt inde ovule er teknisk udfordrende for cytologiske og cytogenetiske analyser på enkelt celle niveau. Især er direkte eller indirekte immunodetektering af cellulære eller nukleare epitoper svækket af dårlig penetration af reagenser inde i plantecellen og single-cell imaging er demised af manglen på optiske klarhed i hel-mount væv. Således har vi udviklet en effektiv metode til at analysere Nucløre organisation og kromatin modifikation ved høj opløsning på enkelt celle i hel-mount indlejrede Arabidopsis frøanlæg. Den er baseret på dissektion og indlejring af faste frøanlæg i et tyndt lag af acrylamid gel på en mikroskopisk slide. Den indlejrede frøanlæg udsættes for kemiske og enzymatiske behandlinger sigter mod at forbedre væv klarhed og permeabilitet til immunfarvning reagenser. Disse behandlinger bevare cellulære og kromatin organisation, DNA og protein-epitoper. Prøverne kan anvendes til forskellige efterfølgende cytologiske analyser, herunder kromatin immunfarvning, fluorescens in situ hybridisering (FISH), og DNA-farvning for heterochromatin analyse. Konfokal laser scanning mikroskopi (CLSM) billeddannelse, med høj opløsning, efterfulgt af 3D rekonstruktion giver mulighed for kvantitative målinger på enkelt-celle opløsning.

Vi giver en robust protokol for forberedelse storstilet og behandling af Arabidopsis frøanlæg egnet til cytologisk farvning i hel-mount. Takket være indlejring, frøanlæg bevare en 3-dimensionel struktur (figur 3). Desuden vævsbehandling, herunder optisk afklaring sætter billedbehandlingsprogrammer subcellulære strukturer på høj opløsning. Figur 4 viser DNA-farvning i hel-mount ovule primordier hvor heterochromatin fremstår som lyse, veldefinerede iøjnefaldende foci...

Watch this Scientific Journal Video about An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis at JoVE.com

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

En effektiv metode til kvantitative, Single-celle Analyse af Kromatin Ændring og nuklear Arkitektur i Whole-Mount frøanlæg i<em&gt; Arabidopsis</em

Vi giver her en effektiv og pålidelig protokol til immunfarvning, Fluorescens in situ hybridisering, DNA-farvning efterfulgt af kvantitativ, høj opløsning billeddannelse i hel-mount Arabidopsis thaliana frøanlæg. Denne metode blev anvendt med succes til at analysere kromatin ændringer og nuklear arkitektur.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Research

JoVE Journal

Biology

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualisering og analyse af mRNA molekyler under anvendelse Fluorescens<em&gt; In Situ</em&gt; Hybridisering i<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Måling rumlige og tidslige Ca<sup&gt; 2+</sup&gt; Signaler i Arabidopsis Planter

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

A Whole Mount<em&gt; In Situ</em&gt; Hybridisering Fremgangsmåde til gastropoden Mollusc<em&gt; stor mosesnegl</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

CUBIC protokol Visualiserer Protein Expression på Single Cell Opløsning i Whole Mount Skin Forberedelser

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Visualisering af 3D hvidt fedtvæv struktur ved hjælp af hele-mount farvning

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

En nem og fleksibel podningsmetode til nøjagtig vurdering af melduginfektionsfænotyper af Arabidopsis og andre planter

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...