Name: zygotic fluorescence recovery after photo-bleaching analysis for chromatin looseness that allows full-term development
Uploaded: 2018-06-12T14:30:00
Description: Watch this Scientific Journal Video about Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development at JoVE.com

Wij danken Satoshi Kishigami, Sayaka Wakayama Hiroaki Nagatomo, Satoshi Kamimura en Kana Kishida voor kritische opmerkingen en technische ondersteuning. Dit werk werd gedeeltelijk gefinancierd door het programma van het ministerie van onderwijs, cultuur, sport, wetenschap en technologie voor het bevorderen van de hervorming van nationale universiteiten M.O.; de Society van Japan voor de bevordering van de wetenschap (16H 02593), Asada Science Foundation en de Stichting van de wetenschap van de Takeda aan T.W. De financiers had geen rol in de studie ontwerp, gegevensverzameling en analyse, besloten tot bekendmaking of voorbereiding van het manuscript.

Deze studie werd uitgevoerd in strikte overeenstemming met de aanbevelingen in de gids voor de zorg en het gebruik van de dieren van het laboratorium van de Universiteit van Yamanashi. Het protocol is goedgekeurd door de Commissie op de ethiek van dier experimenten van de Universiteit van Yamanashi (toestaan nummer: A24-50). Alle operaties werden uitgevoerd onder tribromoethanol narcose en inspanningen werden geleverd om het leed te minimaliseren. Een geïllustreerd overzicht van de procedures en het tijdschema worden we...

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Zoals geopenbaard in deze studie, veroorzaakt de analyse van de zFRAP geen kritische schade aan voldragen ontwikkeling, suggereert dat deze methode is een zeer nuttig instrument om te onthullen van de associatie tussen moleculaire gebeurtenissen en het embryonale ontwikkelings potentieel. Tijdens de herprogrammering, in de twee-cel als staat van ES-cellen, waardoor de differentiële potentieel van deze cellen zo hoog als die van twee cellen fase embryo's wordt, verandert chromatine losheid in die vergelijkbaar zijn met t...

Na de bevruchting, de structuur van de chromatine is dynamisch gewijzigd en zygotic chromatine structuur is dan uiteindelijk gevestigde1,2. Tijdens deze periode, in de vaderlijke pronuclei, is de dominante chromatine proteïne veranderde van tijdje in Histon. De resulterende chromatine is zeer verschillend van die van sperma en eicellen van de vrouw in verschillende punten (bijvoorbeeld, histone variant samenstelling, histone wijziging). Aldus wordt gevormde zygotic chromatine gezien als belangrijk voor de latere ontwikkeling van het embryo. Echter, ondanks pogingen om de details van de structuur van de chromatine zygotic onthullen over lange periodes, methoden voor de beoordeling van de kwaliteit van zygoten of te voorspellen hun voldragen ontwikkeling in het stadium van een cel door het analyseren van de structuur van de chromatine nooit geweest opgericht.
In de vorige studie, is het ontdekt dat zygoten een uiterst losgedraaide chromatine structuur3 hebben. Op dit moment de losheid van de chromatine of openheid wordt verondersteld te zijn een belangrijke factor voor celdifferentiatie potentieel in embryonale stamcellen (ES) cellen4. ES-cellen homogeniteit in de natuur geen vertonen, maar zijn nogal heterogene; in de kolonies van de cel van de ES verwerven sommige Transient een hogere differentiatie mogelijkheden vergelijkbaar met blastomeren van twee cellen fase embryo's. Tijdens deze overgang in de twee-cel als staat cellen chromatine losheid in ES verandert in wat vergelijkbaar met twee-cel fase embryo's5 is. Zo lijkt de chromatine losheid van belang voor celdifferentiatie potentieel en het is mogelijk dat uitgebreid open chromatine in zygoten is handig voor de evaluatie van zygotic ontwikkelings potentieel.
Levende imaging is een krachtig hulpmiddel waarmee voor de analyse van de moleculaire gebeurtenissen die tijdens de ribben omdat deze methode geschikt is voor verdere ontwikkeling en zelfs voldragen ontwikkeling6. Als een van de levende imaging methoden, is FRAP analyse gebruikt om te onderzoeken van de chromatine losheid in voor inplanting embryo's en ES cellen3,4,5. Als zygotic chromatine losheid kan worden geanalyseerd zonder een schadelijk effect op voldragen ontwikkeling door FRAP analyse, kan het zijn een waardevol instrument voor de beoordeling van de kwaliteit van de embryo's in het stadium van de één-cel. Echter zijn de effecten op voldragen ontwikkeling door deze experimentele methode niet onderzocht. Onlangs, een experimenteel systeem met behulp van FRAP te evalueren zygotic chromatine losheid werd ontwikkeld. Omdat dit een nieuwe waarnemingssysteem voor zygoten was, werd het genoemd als zygotic FRAP (zFRAP). zFRAP beïnvloedde niet kritisch voldragen ontwikkeling en is elders7gemeld. In dit verslag, wordt het protocol voor deze experimentele methode beschreven.

<table border="0" cellpadding="0" cellspacing="0" width="1022">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">Confocal laser scaning microscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">FV1200</td>
			<td style="width:435px;">FV1000 can be also used for FRAP analysis (reference #7)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Thermo plate</td>
			<td style="width:208px;">Tokai Hit</td>
			<td style="width:119px;">TP-110RH26</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Inverted microscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Micro manupilator</td>
			<td style="width:208px;">Narishige</td>
			<td style="width:119px;">MMO-202ND</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Pieze Micro Micromanipulator</td>
			<td style="width:208px;">Prime tech</td>
			<td style="width:119px;">PMAS-CT150</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">35 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351008</td>
			<td>35 x 100 mm style; for IVF</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">60 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351007</td>
			<td>60 x 15 mm style; for embryo culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">50 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351006</td>
			<td>50 x 9 mm style; for manipulation on the stage</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">50 mm glass bottom dish</td>
			<td style="width:208px;">Matsunami</td>
			<td style="width:119px;">D910400</td>
			<td>50 mm dish, 27 mm &#966; hole size; for FRAP analysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Mineral oil</td>
			<td style="width:208px;">Organic spceiality chemicals</td>
			<td style="width:119px;">625071</td>
			<td>For FRAP analysis on the glass bottom dishes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Mineral oil</td>
			<td style="width:208px;">Sigma</td>
			<td style="width:119px;">M8410-1L</td>
			<td>For IVF and embryo culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">glass capillary</td>
			<td style="width:208px;">Drummond</td>
			<td style="width:119px;">1-000-1000</td>
			<td>For handling mouse zygotes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Borosilicate glass</td>
			<td style="width:208px;">Prime tech</td>
			<td style="width:119px;">B100-75-10-PT</td>
			<td>For microinjection of mRNA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">Micropipett puller</td>
			<td style="width:208px;">Sutter instrument</td>
			<td style="width:119px;">P-97/IVF</td>
			<td style="width:435px;">For preparation of the injection or holding neadle (out side diameter: 80 &#181;m, inner diameter: 10 &#181;m)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Microforge</td>
			<td style="width:208px;">&#160;Narishige</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">mMESSAGE MACHINE SP6 Transcription kit</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM1340</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">Poly (A) tailing kit</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM1350</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">PVP solution 10% (PVP-HTF)</td>
			<td style="width:208px;">IrvineSceientific</td>
			<td style="width:119px;">99311</td>
			<td>HEPES buffered HTF containing 10% PVP</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Sodium HEPES</td>
			<td style="width:208px;">Sigma</td>
			<td style="width:119px;">H3784</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">pTOPO eGFP-H2B</td>
			<td style="width:208px;"></td>
			<td style="width:119px;"></td>
			<td>Template plasmid for eGFP-H2B mRNA (reference #6)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">NorthernMax Gly Sample loading Dye&#160;</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM8551</td>
			<td>For electrophoresis of in vitro transcribed mRNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Phenol chloroform isamyl alcohol</td>
			<td style="width:208px;">nacalai tesque</td>
			<td style="width:119px;">25970-14</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Chloroform</td>
			<td style="width:208px;">nacalai tesque</td>
			<td style="width:119px;">&#160;08401-65</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Not I</td>
			<td style="width:208px;">TOYOBO</td>
			<td style="width:119px;">NOT-111X</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Ethachinmate</td>
			<td style="width:208px;">WAKO</td>
			<td style="width:119px;">318-01793</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">3664 Otical power meter</td>
			<td style="width:208px;">Hioki</td>
			<td style="width:119px;">3664</td>
			<td>&#160;Power meter for laser power</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">Stereomicmicroscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">SZX16</td>
			<td></td>
		</tr>
	</tbody>
</table>

zygotic fluorescence recovery after photo-bleaching analysis for chromatin looseness that allows full-term development

Levende imaging is een krachtig hulpmiddel waarmee voor de analyse van de moleculaire gebeurtenissen die tijdens de ribben. Onlangs, chromatine losheid of openheid is aangetoond te worden betrokken in het potentieel van de celdifferentiatie van pluripotente embryonale stamcellen. Eerder werd gemeld dat in vergelijking met embryonale stamcellen, zygoten haven een uiterst losgedraaide chromatine-structuur, die haar relatie met hun totipotent duiden. Echter, tot nu toe, het niet is beantwoord of de structuur van deze uiterst losgemaakt/open chromatine belangrijk voor de embryonale ontwikkelings potentieel is. In de huidige studie, om te onderzoeken van deze hypothese, werd een experimenteel systeem welke zygoten die werden geanalyseerd door fluorescentie herstel na foto-bleken zich tot termijn zonder significante schade ontwikkelen kan ontwikkeld. Nog belangrijker is, moet dit experimenteel systeem alleen een kachel van thermos-plaat naast een confocale laser scanning microscoop. De bevindingen van deze studie suggereren dat herstel van de fluorescentie na foto-bleken analyse (FRAP) analyse kan worden gebruikt om te onderzoeken of de moleculaire gebeurtenissen in zygotic chromatine belangrijk voor de ontwikkeling van de voldragen zijn.

zFRAP analyse met eGFP-H2B
MRNA codering eGFP-H2B, die wordt gezien als een verschoven band door poly een tailing (figuur 2A veroorzaakt), goed geproduceerd en werd geïnjecteerd in het cytoplasma van zygoten met een 2e polar lichaam 1-3 uur na inseminatie (figuur 2B). Acht tot 12 uur na de inseminatie, zygoten met 2 pronuclei tonen de expressie van eGFP-H2B werden verzame...

Watch this Scientific Journal Video about Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development at JoVE.com

Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development

Chromatine losheid lijkt te worden betrokken in het ontwikkelings potentieel van blastomeren. Het is echter niet bekend of chromatine losheid kan worden gebruikt als een betrouwbare index voor de ontwikkelings potentieel voor embryo's. Hier, is een experimenteel systeem waarin chromatine losheid-geëvalueerd zygoten tot volledige termijn ontwikkelen kunnen beschreven.

Zygotic fluorescentie herstel na het ontkleuren aangebracht van foto analyse voor chromatine losheid waarmee voldragen ontwikkeling

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been ...

The overall goal of this zygotic FRAP or zFRAP is to examine the association between zygotic chromatin looseness and embryonic development potential to term. This method can help answer key questions in the developmental biology field, such as embryonic development. The main advantage of this technique is that it allows the analyzed zygote to develop to full term without significant damage.Visual demonstration of this method is critical as the zFRAP analysis steps are difficult to learn because the experimental condition setting and operation for this analysis are very complex. After preparing eGFP-H2B RNA according to the text protocol, use nuclease-free water to dilute it to 250 nanograms per microliter. In a 60-millimeter dish, place two to three drops each of PVP-HTF, five to six drops of Hepes-buffered CZB, and eGFP-H2B mRNA.Then, use mineral oil to cover the drops. Using a pipette puller, pull borosilicate glass. After breaking the tip, use a microforge to bend the glass pipette to around 30 degrees.Prior to microinjection, confirm that the plate heater on the stage of the micromanipulator is off to prevent killing the zygotes during mRNA injection. Then, using 10%PVP-HTF, wash and coat the inside of the injection needles. Next from the tips fill the glass capillaries with the diluted eGFP-H2B mRNA.Collect the fertilized zygotes with a second polar body, and transfer 20 to 25 of them on to the chamber of the microscope stage. Then, with a holding pipette, hold the zygote and break the zona pellucida and the cytosolic membrane with a piezo drive. Inject about 10 picoliters of mRNA into the cytoplasm of each zygote.Complete the injections within 10 minutes to prevent damage caused by culturing the zygotes outside of the incubator. 10 minutes after microinjection, wash the zygotes in at least three drops of CZB and culture them at 38 degrees Celsius until FRAP analysis. One hour prior to zFRAP analysis, turn on the laser and hot plate attached to the stage of the confocal microscope.Set the temperature to 38 degrees Celsius. On a 60-millimeter glass-bottomed dish, pipette six to 10 microliters of Hepes-buffered CZB medium and cover it with mineral oil. Then, warm the dish on the heater until zygote collection.To make it easy to find the zygote with the confocal microscope, it is important to put the drops throughout the center of the dish. Eight to 12 hours after insemination, collect five to 10 eGFP-H2B expressing zygotes and transfer them to a six to 10-microliter drop of pre-warmed Hepes-buffered CZB medium. To perform a precise quantitative analysis it is important to put the zygote toward the center of the Hepes-buffered CZB drops.Set the bleaching and imaging conditions according to the manufacturer's instructions. For example, under Acquisition Setting, set the scanning speed to four microseconds per pixel and the size to five hundred and twelve. Increase the digital zoom to five, open the DyeList"and choose eGFP"Then, set the observation time to one point six seconds and the number of pictures to 12.Next, start the Stimulus Setting"panel and on UseScanner"click Main"Set the scanning speed to 10 microseconds per pixel. Click Activation in Series"Set PreActivation"to three frames and activation time to five seconds. Then, click Focus x2"and click Stop"Under the stimulus setting panel click Clip Rect"Set the ROI to 40 by 40 pixels.Click Time"and XY"Then, use an optical power meter to access the laser power. It is important to have the lights turned off when measuring the laser. After adjusting the laser gage to set the bleaching and imaging laser power, on the toolbar click Live"and then start Live Plot"Place the oil on the objective lens.Set the chamber on the stage and then find the zygotes in the drops. After finding zygotes, determine the ROI position by clicking Focus x2"And then in the Live Plot"window adjust the HV power gage for the eGFP"channel fluorescence intensity to around 2, 000. To start the zFRAP analysis, click XY"After FRAP analysis, click Series Done"Obtain the FRAP analyzed data, then click on Save As"to save the file 2D View image"as a preferred named file.Use Copy"ROI and Paste"ROI to prepare a reference region and to background respectively. Next, select ROI, REF, and BG, and then in the 2D View"image window, click Series Analysis"Obtain RoFRAP data and then in the Live Plot"window click the Save"button. Carry out data calculations according to the text protocol.Eight to 12 hours after insemination, zygotes with two pronucleai showing the expression of eGFP-H2B"were collected and subjected to zFRAP analysis. After bleaching, the intensity of the eGFP-H2B fluorescent signal recovered slightly because of the inflow of eGFP-H2B into the ROI from the unbleached area in the nucleoplasm. Recovery curves reveal that male pronucleai recovered more quickly and with more mobile fractions than female pronucleai.As demonstrated in these images and graph, after zFRAP analysis, washed and cultured zygotes in CZB medium developed to the blastocyst stage without significant damage. To analyze the full term development, embryos at the two-cell stage were transferred into the ovoducts of pseudo-pregnant mothers. As reported in this table, the birth rate of zFRAP analyzed embryos was slightly, but significantly lower than that of intact embryos but was the same as that of the no zFRAP control.In addition, the pups derived from zFRAP analyzed zygotes seemed healthy and fully developed. While attempting this procedure, it's important to remember to wait for the other expansion. All budget objectorance to avoid focus drift.After watching this video, you should have a good understanding on how to analyze chromatin looseness in zygotes.

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Meten Eiwit Stabiliteit in Living zebravis embryo&#39;s met behulp van fluorescentie Decay Na Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Genoom-brede Snapshot van chromatine regelgevers en staten in<em&gt; Xenopus</em&gt; Embryo&#39;s van Chip-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Protocollen voor het verkrijgen Zygotic en somatische embryo&#39;s voor het bestuderen van de verordening van de vroege embryo ontwikkeling in de Model Legume<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Analyse van Cardiomyocyte Development behulp Immuunfluorescentie in Embryonale Muis Hart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Analyse van de zebravis Larven skeletspieren Integriteit met Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

Isolatie en karakterisering van de Muis Antral Eicellen Gebaseerd op Hergroepering van nucleoli chromatine Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Analyse van de zebravis Kidney Development met Time-lapse Imaging Met behulp van een dissectie microscoop Uitgerust voor Optische-Sectie

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Met behulp van Light Sheet fluorescentiemicroscopie met Afbeelding zebravis Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Ontwikkeling van een<em&gt; In Vitro</em&gt; Assay om contractiele functie van mesenchymale Cellen die Onderging-mesenchymale transitie Evalueer

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computationele analyse van de Caenorhabditis elegans Germline te bestuderen van de distributie van kernen, eiwitten en het cytoskelet

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...