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

Vi tackar Satoshi Kishigami, Sayaka Wakayama, Hiroaki Nagatomo, Satoshi Kamimura och Kana Kishida för kritiska synpunkter och tekniska stöd. Detta arbete finansierades delvis av ministeriet för utbildning, kultur, sport, vetenskap och teknik programmet för att främja reformen av nationella universitet att M.O.; Japan Society för främjande av vetenskap (16H 02593), Asada Science Foundation och stiftelsen Takeda vetenskap att T.W. Finansiärerna hade ingen roll i studiedesign, datainsamling och analys, beslut om att offentliggöra eller beredning av manuskriptet.

Denna studie utfördes i strikt överensstämmelse med rekommendationerna i Guide för skötsel och användning av laboratoriedjur av de universitet i Yamanashi. Protokollet godkändes av kommittén för etik av djur experimenten av de universitet i Yamanashi (tillåta nummer: A24-50). Alla operationer utfördes under tribromoethanol anestesi, och alla ansträngningar gjordes att minimera lidande. En illustrerad översikt av förfaranden och tidsplan visas i figur 1 och tabell 1</str...

<ol>
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Som framkommit i denna studie, orsakar den zFRAP analysen inte kritiska skador till fullgångna utveckling, tyder på att denna metod är ett mycket användbart verktyg för att avslöja sambandet mellan molekylära händelser och den embryonala utvecklingspotentialen. Under omprogrammering, in i två-cellen som stat av ES-celler, som differentiell potentialen i dessa celler blir lika hög som för två-cellstadie embryon, ändras kromatin glapp in i det jämförbara med två-cellstadie embryon5. ...

Efter befruktningen, kromatinstruktur ändras dynamiskt, och zygotic kromatinstruktur är sedan så småningom etablerade1,2. Under denna period, i Faderns pronuclei, ändras proteinet dominerande kromatin från Protamin till Histon. Det resulterande kromatinet skiljer sig extremt från spermier och kvinnliga oocyter i flera punkter (t.ex., Histon variant sammansättning, Histon modifiering). Således tros bildade zygotic kromatin vara viktigt för efterföljande embryonal utveckling. Men trots ansträngningar att avslöja detaljerna i zygotic kromatinstrukturen under långa perioder, har metoder att utvärdera kvaliteten på zygoter eller att förutsäga deras fullgången utveckling i en-cellstadie genom att analysera deras kromatinstruktur aldrig varit etablerade.
I den tidigare studien upptäcks det att zygoter har en extremt lossade kromatin struktur3. För närvarande, tros kromatin glapp eller öppenhet vara en viktig faktor för celldifferentiering potential i embryonala stamceller (ES) celler4. ES-celler uppvisar inte homogenitet i naturen, men är ganska heterogen; i ES cell kolonier få vissa övergående en högre differentiering potential jämförbar med blastomerer två-cellstadie embryon. Under denna övergång in i två-cellen som stat, kromatin glapp i ES-celler förändringar i vad som är jämförbara med två-cellstadie embryon5. Kromatin glapp verkar således vara viktigt för celldifferentiering potential och det är möjligt att i stor utsträckning öppna kromatin i zygoter är användbart för utvärdering av zygotic utvecklingspotential.
Levande bildbehandling är ett kraftfullt verktyg som möjliggör analys av molekylära händelser under ontogenin eftersom denna metod möjliggör framtida utveckling och även fullgångna utveckling6. Som en av de levande avbildningsmetoder, har FRAP analys använts för att undersöka kromatin glapp i preimplantatorisk embryon och ES celler3,4,5. Om zygotic kromatin glapp kan analyseras utan en skadlig effekt på fullgångna utveckling av FRAP analys, kan det vara ett värdefullt verktyg för utvärdering av kvaliteten på embryon i en-cellstadie. Effekterna på fullgångna utvecklingen av denna experimentella metod har dock inte undersökts. Nyligen, en experimentell system använder FRAP för att utvärdera zygotic kromatin glapp utvecklades. Eftersom detta var ett nytt observationssystem för zygoter, var det betecknas som zygotic FRAP (zFRAP). zFRAP påverkade inte kritiskt fullgången utveckling och har varit rapporterade någon annanstans7. I denna rapport beskrivs i protokollet av den experimentella metoden.

<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

Levande bildbehandling är ett kraftfullt verktyg som möjliggör analys av molekylära händelser under ontogenin. Nyligen, kromatin glapp eller öppenhet har visat sig vara inblandade i celldifferentiering potential av pluripotenta embryonala stamceller. Det rapporterades tidigare som jämfört med embryonala stamceller, zygoter harbor en extremt lossade kromatinstruktur, tyder på associationen med deras totipotency. Men fram till nu, har det inte behandlats om denna extremt lossade/öppna kromatinstruktur är viktigt för embryonala utvecklingspotentialen. I den aktuella studien, för att pröva denna hypotes, utvecklades en experimentell system i vilka zygoter som analyserades av fluorescens återhämtning efter foto-blekning kan utvecklas till sikt utan betydande skada. Allt behöver detta experimentella system endast en termos-plattan värmare förutom en confocal laser skanning Mikroskop. Resultaten av denna studie föreslår att fluorescens återhämtning efter foto-blekning analys (FRAP) analys kan användas för att undersöka om de molekylära händelserna i zygotic kromatin är viktigt för fullgångna utveckling.

zFRAP analys med andra-H2B
Korrekt producerad injicerades mRNA kodning andra-H2B, som ses som ett skiftat band orsakas av poly en tailing (figur 2A), i cytoplasman i zygoter med en 2: a polar kropp på 1-3 h efter insemination (figur 2B). Åtta till 12 h efter insemination, zygoter med 2 pronuclei visar uttrycket av andra-H2B samlades och utsätts för zFRAP analys (<stron...

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

Kromatin glapp verkar vara inblandade i den utvecklande potentialen av blastomerer. Det är dock inte känt om kromatin glapp kan användas som en pålitlig index för den utvecklande potentialen för embryon. Här, har en experimentell system där kromatin glapp-utvärderas zygoter kan utvecklas till full sikt beskrivits.

Zygotic fluorescens återhämtning efter foto-blekning analys för kromatin glapp som möjliggör fullgången utveckling

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

Mätning Protein Stabilitet i Living Zebrafish embryon Använda Fluorescens Decay Efter 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

Genomvid ögonblicksbild av kromatin tillsynsmyndigheter och stater i<em&gt; Xenopus</em&gt; Embryon från 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

Protokoll för att erhålla Zygotic och somatiska embryon för att studera reglering av tidig embryoutveckling i Model baljväxter<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

Analys av cardiomyocyte Development använder Immunfluorescens i embryonal Mouse Heart

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

Analys av Zebrafish Larver skelettmuskulatur Integrity med 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

Isolering och karakterisering av mus Antral oocyter Baserat på Nukleolärt Chromatin 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

Analys av zebrafisk njure utveckling med Time-lapse avbildning med ett dissektionsmikroskop Utrustad för optisk Sektione

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

Använda ljus Sheet fluorescensmikroskopi till bild Zebrafish Eye utveckling

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

Utveckling av en<em&gt; In Vitro</em&gt; Analys för att utvärdera kontraktila funktion av mesenkymala celler som genomgick Epithelial-Mesenkymala Övergångs

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

Computational analys av den Caenorhabditis elegans könsceller att studera fördelningen av atomkärnor, proteiner och cytoskelettet

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