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).

Fremgangsmåten er beskrevet i arbeidsflyten i figur 1, og oppsett for disseksjon og innstøping av vev er presentert i figur 2.. En. Tissue Fixation <ol><li> Samle 20-30 carpels i en mikro rør som inneholder rykende BVO fiksativ buffer på is. </li><li> Fest vev 30 min med forsiktig risting ved romtemperatur. </li><li> Spinn rør som inneholder de carpels i fiksativ i en bordmikrosentrifuge 1 min ved 400 x g. </li><li> Fjern forsiktig den fiksativ buffer...

<ol>
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I blomsterplanter, er den kvinnelige reproduktive avstamning omgitt av flere cellelag inkludert nucellus og ovule teguments, dermed rende cytologisk flekker i hel-mount teknisk utfordrende. Her presenteres en effektiv protokoll som muliggjør fremstilling og bearbeiding av et stort antall ovuler egnet for cytologisk farging som farging, DNA farging og fluorescens in situ hybridisering i hel-festet. Vi hell brukt det for analyse av den kvinnelige reproduktive germline i Arabidopsis 21,22. Den...

I blomstrende planter, begynner etablering av reproduktive linjene med differensiering av SMCS, kvinnelig MMC og mannlige microspore mor celle. MMC utvikler seg fra en sub-epidermal nucellar celle ved den distale enden av ovule primordium, og microspore mor celle utvikler seg fra sporogenous vev i anther locule, som ligger dypt inne i blomster organer en. SMCS gjennomgå meiose for å produsere haploide sporer, som deretter gir opphav til de gametophytes ved mitose. Den kvinnelige gametofytt, eller embryo sac, består av ett egg celle, en sentral celle, to synergids og tre antipodals. Den mannlige gametofytt eller pollen, er sammensatt av en vegetativ celle og to sædceller. Mens den mannlige gametofytt fortsatt en relativt tilgjengelig objekt-of-studien, er den kvinnelige gametofytt innebygd i ovule, selv vedlagt i blomster carpel, og dermed stiller konkrete utfordringer til molekylære og cytologiske analyser. Nylig har imidlertid laserassistertmicrodissection tilbudt en elegant løsning slik transcriptomic analyser i MMC og kvinnelige gametophytic celler 2-4. I tillegg til kandidat genekspresjon analyser, for eksempel ved bruk RNA in situ hybridisering eller reporter gen analyser, cytologiske analyser lar undersøke dynamikken i endogene cellulære komponenter ved hjelp av spesifikke direkte cellefarging eller indirekte farging. Spesielt, cytogenetisk flekker ved hjelp av FISH og DNA farging, sammen med farging av kromatin endringer eller kromatin komponenter er sentrale innfallsvinkler for å belyse kromatin dynamikk og atom organisasjon i Arabidopsis fem. Vanligvis medfører meiose spesifikke kromosom dynamikk som har blitt godt undersøkt i anlegget mannlig meiocytes 6,7; videre i stor skala, celle-spesifikke kromatin omorganisering, som sannsynligvis gjenspeiler dynamisk epigenetisk reprogrammering har blitt beskrevet under pollen utvikling 8-10. I motsetning til dette, på grunnav den relative utilgjengelighet av den kvinnelige meiocyte og gametofytt, disse undersøkelsene forblir teknisk vanskelig å påføre, og krever ofte snitte eller manuell disseksjon og enzymatisk fordøyelse (se nedenfor). I tillegg er det utbredt mangel på optisk klarhet i hel-mount et hinder for høyoppløselig avbildning av kjønnsceller i intakte ovules. En klassisk metode for cytologisk analyse av kromosom organisasjon i hel-mount ovules bruker Feulgen er flekker 11-13. Det omfatter syrehydrolyse (ved bruk av hypoklorsyre) av DNA som resulterer i protein denaturering og dermed fører til ødeleggelse av kromatin struktur. Alternativt kan observeres kromosom organisasjon i kvinnelige meiocytes og gametophytic celler ved hjelp DAPI farging og farging på semi-tynne snitt eller dissekert embryo sekker og MMC (for eksempel se 14-18). Det er imidlertid klart, manuell disseksjon og seksjonering kan være arbeidskrevende oghindrer på kvalitativ og kvantitativ analyse av et stort antall kromatin epitoper. Her gir vi en effektiv protokoll for å forberede et stort antall Arabidopsis ovules egnet for en rekke nedstrøms cytologisk flekker i hel-mount. I korte trekk, er blomsterknopper inkuberes i en fiksativ løsning, er rader med ovules dissekert fra carpel og innebygd i akrylamid på lysbildet som gjøres for pollen meiocytes 19,20. De innebygde ovuler er ytterligere fjernet og fiksert i metanol, etanol, og xylen før cellevegg fordøyelse og permeabilization. Mulige variasjoner av denne fremgangsmåten er omtalt. Prøvene kan deretter bli brukt for DNA beising, farging, og fisk. Preparatet modus er effektiv og gjør det mulig ved parallell eksperimentelle oppsett (opp til 16 slides, kan fremstilles på en dag etter ulike nedstrøms-analyse). Behandlingene er beskrevet aktiver homogene signaler i hel-mount og godt bevart histologisk, mobilnettet,og kjernefysiske organisasjon i kjønnsceller og omkringliggende nucellar celler som gagner kvalitative og kvantitative sammenligninger mellom celletyper. Kalibrert, CLSM-baserte høy oppløsning avbildning etterfulgt av 3-dimensjonale rekonstruksjon muliggjør meningsfylte kvantitative målinger av fluorescerende signaler. Vi hell brukt denne prosedyren for å analysere kromatin dynamikk i differensierende MMC 21 og utvikle kvinnelige gametofytt 22; vi presenterer her representative resultatene av heterochromatin analyse, kromatin farging, GFP farging og FISH i hel-mount ovules. Vi tror videre at vår protokoll vil være egnet for andre plantevev 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 blomsterplanter, er det somatiske-til-reproduktiv celle skjebne overgang preget av spesifikasjonen av spore mor celler (SMCS) i floral organer i voksen plante. Den kvinnelige SMC (megaspore mor celle, MMC) skiller i ovule primordium og gjennomgår meiose. Den valgte haploid megaspore deretter gjennomgår mitose å danne flercellet kvinnelige gametofytt, noe som vil gi opphav til kjønnscellene, eggcellen og sentral celle, sammen med tilbehør celler. Den begrensede tilgjengeligheten av MMC, meiocyte og kvinnelige gametofytt inne ovule er teknisk utfordrende for cytologisk og cytogenetiske analyser på celle-nivå. Spesielt er direkte eller indirekte immuno-deteksjons av cellulære eller atom epitoper svekket av dårlig penetrering av reagensene inne i plantecellen, og enkeltcelle-avbildning er demised av mangelen på optisk klarhet i hel-typen vev. Således, har vi utviklet en effektiv metode for å analysere Nucløret organisasjon og kromatin modifisering i høy oppløsning på én celle i hel-mount innebygde Arabidopsis ovules. Den er basert på disseksjon og innstøping av faste ovuler i et tynt lag av akrylamid gel på et mikroskopisk lysbilde. De innebygde ovules er utsatt for kjemiske og enzymatiske behandlinger som tar sikte på å bedre vev klarhet og permeabilitet til farging reagenser. Disse behandlingene bevare cellulære og kromatin organisasjon, DNA og protein epitoper. Prøvene kan brukes for forskjellige nedstrøms cytologiske analyser, inkludert kromatin farging, fluorescens in situ hybridisering (FISH), og DNA-farging for heterochromatin analyse. Konfokal laser scanning mikroskopi (CLSM) bildebehandling, med høy oppløsning, etterfulgt av 3D rekonstruksjon åpner for kvantitative målinger ved encellede oppløsning.

Vi tilbyr en robust protokoll for storskala utarbeidelse og behandling av Arabidopsis ovules egnet for cytologisk farging i hel-mount. Takket være innebygging, de ovules beholde en 3-dimensjonal struktur (figur 3). Videre vevet foredling herunder optisk avklaring lar bildebehandling subcellulære strukturer i høy oppløsning. Figur 4 viser DNA flekker i hel-mount ovule primordia hvor heterochromatin fremstår som lyse, godt definert iøynefallende fokus (ingen deconvolution b...

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 for kvantitativ, Single-celle Analyse av Kromatin Modifikasjon og Nuclear Arkitektur i hel-mount ovules i<em&gt; Arabidopsis</em

Vi gir her en effektiv og pålitelig protokoll for farging, fluorescens in situ hybridisering, DNA farging fulgt av kvantitativ, bildebehandling med høy oppløsning i hel-mount Arabidopsis thaliana ovules. Denne metoden ble brukt for å analysere kromatin modifikasjoner og atom 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 av mRNA molekyler med 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

Måle romlig og tidsmessig 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 Metode for Gastropod bløtdyr<em&gt; stor damsnegl</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 Protocol Visualiserer Protein Expression på enkelt celle oppløsning i Hele Mount hud preparater

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 av 3D hvit fettvev struktur med hele-mount flekker

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 enkel og fleksibel inokulasjonsmetode for nøyaktig vurdering av pulverformig mugginfeksjon Fenotyper av 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 ...