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 takker Satoshi Kishigami, Sayaka Wakayama, Hiroaki Nagatomo, Satoshi Kamimura og Kana Kishida for kritiske kommentarer og teknisk support. Dette arbejde blev delvist finansieret af Ministeriet for uddannelse, kultur, sport, videnskab og teknologi-program for at fremme reformen af nationale universiteter til M.O.; Japan-samfund til fremme af videnskab (16H 02593), Asada Science Foundation og Takeda Science Foundation til T.W. Finansieringskilderne spillede ingen rolle i undersøgelse design, dataindsamling og analyse, beslutningen om at offentliggøre eller forberedelse af manuskriptet.

Denne undersøgelse blev udført i nøje overensstemmelse med anbefalinger i vejledningen for pleje og brugen af forsøgsdyr i Yamanashi Universitet. Protokollen blev godkendt af Udvalget om etik af dyr eksperimenter på Universitet Yamanashi (tillade nummer: A24-50). Alle operationer blev udført under tribromoethanol anæstesi, og alle bestræbelser blev gjort til at minimere lidelse. En illustreret oversigt over de procedurer og tidsplan er vist i figur 1 og tabel 1, henh...

<ol>
	<li>Burton, A., Torres-Padilla, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+reprogramming+and+development:+a+unique+heterochromatin+organization+in+the+preimplantation+mouse+embryo.">Epigenetic reprogramming and development: a unique heterochromatin organization in the preimplantation mouse embryo.</a> Brief Funct Genomics. 9, 444-454 (2010).</li><li>Okada, Y., Yamaguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+modifications+and+reprogramming+in+paternal+pronucleus:+sperm,+preimplantation+embryo,+and+beyond.">Epigenetic modifications and reprogramming in paternal pronucleus: sperm, preimplantation embryo, and beyond.</a> Cell Mol Life Sci. 74, 1957-1967 (2017).</li><li>Ooga, M., Fulka, H., Hashimoto, S., Suzuki, M. G., Aoki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chromatin+structure+in+mouse+preimplantation+embryos+by+fluorescent+recovery+after+photobleaching.">Analysis of chromatin structure in mouse preimplantation embryos by fluorescent recovery after photobleaching.</a> Epigenetics. 11, 85-94 (2016).</li><li>Meshorer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdynamic+plasticity+of+chromatin+proteins+in+pluripotent+embryonic+stem+cells.">Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells.</a> Dev Cell. 10, 105-116 (2006).</li><li>Boskovic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+chromatin+mobility+supports+totipotency+and+precedes+pluripotency+in+vivo.">Higher chromatin mobility supports totipotency and precedes pluripotency in vivo.</a> Genes Dev. 28, 1042-1047 (2014).</li><li>Yamagata, K., Ueda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live-cell+imaging+of+mammalian+preimplantation+development+and+derivation+process+of+pluripotent+stem+cells+from+the+embryos.">Long-term live-cell imaging of mammalian preimplantation development and derivation process of pluripotent stem cells from the embryos.</a> Dev Growth Differ. 55, 378-389 (2013).</li><li>Ooga, M., Wakayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+analysis+of+chromatin+looseness+in+mouse+zygotes+that+allows+full-term+development.">FRAP analysis of chromatin looseness in mouse zygotes that allows full-term development.</a> PLoS One. 12, e0178255 (2017).</li><li>Quinn, P., Begley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+human+seminal+plasma+and+mouse+accessory+gland+extracts+on+mouse+fertilization+in+vitro.">Effect of human seminal plasma and mouse accessory gland extracts on mouse fertilization in vitro.</a> Australian journal of biological sciences. 37, 147-152 (1984).</li><li>Chatot, C. L., Lewis, J. L., Torres, I., Ziomek, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+1-cell+embryos+from+different+strains+of+mice+in+CZB+medium.">Development of 1-cell embryos from different strains of mice in CZB medium.</a> Biology of reproduction. 42, 432-440 (1990).</li><li>Bae, J., Sung, B. H., Cho, I. H., Song, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F-actin-dependent+regulation+of+NESH+dynamics+in+rat+hippocampal+neurons.">F-actin-dependent regulation of NESH dynamics in rat hippocampal neurons.</a> PLoS One. 7, e34514 (2012).</li><li>Dieteren, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+mitochondrial+translation+differently+affects+the+live+cell+dynamics+of+complex+I+subunits.">Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits.</a> Biochim Biophys Acta. 1807, 1624-1633 (2011).</li><li>Subramanian, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2A.Z+acidic+patch+couples+chromatin+dynamics+to+regulation+of+gene+expression+programs+during+ESC+differentiation.">H2A.Z acidic patch couples chromatin dynamics to regulation of gene expression programs during ESC differentiation.</a> PLoS Genet. 9, e1003725 (2013).</li><li>Hayashi-Takanaka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+epigenetic+histone+modifications+in+single+cells+using+Fab-based+live+endogenous+modification+labeling.">Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.</a> Nucleic Acids Res. 39, 6475-6488 (2011).</li><li>Yamazaki, T., Yamagata, K., Baba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+and+retrospective+analysis+of+DNA+methylation+in+mouse+preimplantation+embryos+by+live+cell+imaging.">Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging.</a> Dev Biol. 304, 409-419 (2007).</li><li>Puschendorf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRC1+and+Suv39h+specify+parental+asymmetry+at+constitutive+heterochromatin+in+early+mouse+embryos.">PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos.</a> Nat Genet. 40, 411-420 (2008).</li></ol>

Som åbenbaret i denne undersøgelse, forårsage zFRAP analyse ikke kritisk skade fuldbårne udvikling, tyder på, denne metode er et meget nyttigt redskab til at afsløre sammenhængen mellem molekylære begivenheder og embryonale udviklingsmæssige potentiale. Under omprogrammering, ind i to-cellen som stat af ES-celler, hvorved differential potentialet i disse celler bliver så stor som to-celle stadium embryoner, ændringer kromatin løshed ind, at sammenlignelige med to-celle stadium embryoner5</su...

Efter befrugtningen, kromatin struktur ændres dynamisk og zygotisk kromatin struktur er derefter i sidste ende etableret1,2. I denne periode, faderlige pronuclei, er dominerende kromatin protein ændret fra protamine til Histon. Den resulterende kromatin er meget forskellig fra sperms og kvindelige oocyter i flere punkter (fx, Histon variant sammensætning, Histon ændring). Således menes dannet zygotisk kromatin at være vigtig for efterfølgende embryonale udvikling. Trods bestræbelser på at afsløre detaljer om zygotisk kromatin struktur over lange perioder, har metoder til at vurdere kvaliteten af zygotes eller at forudsige deres fuldbårne udvikling på én-celle stadium ved at analysere deres kromatin struktur dog aldrig været etableret.
I den tidligere undersøgelse, er det opdaget, at zygotes har en ekstremt løsnede kromatin struktur3. I øjeblikket, menes kromatin løshed eller åbenhed at være en vigtig faktor for Celledifferentiering potentielle i embryonale stamceller (ES) celler4. ES-celler udstille ikke homogenitet i naturen, men er temmelig heterogene; i ES celle kolonier erhverve nogle forbigående en højere differentiering potentiale svarende til blastomeres to-celle stadium embryoner. Under denne overgang ind i to-cellen som stat celler kromatin løshed i ES ændringer i hvad er sammenlignelige med to-celle stadium embryoner5. Kromatin løshed synes således at være vigtig for cellulære differentiering potentiale og det er muligt, at omfattende åbne kromatin i zygotes er nyttige til vurdering af zygotisk udviklingsmæssige potentiale.
Live imaging er et kraftfuldt værktøj, der giver mulighed for analyse af molekylære begivenheder under ontogenesis, da denne metode giver mulighed for efterfølgende udvikling og endda fuldbårne udvikling6. Som en af de live imaging metoder, er FRAP analyse blevet brugt til at undersøge kromatin løshed i præimplantations embryoner og ES celler3,4,5. Hvis zygotisk kromatin løshed kan analyseres uden en skadelig virkning på fuldbårne udvikling af FRAP analyse, kan det være et værdifuldt redskab til evaluering af kvaliteten af embryoner på én-celle stadium. Effekter på fuld-sigt udvikling af denne eksperimentelle metode er dog ikke undersøgt. For nylig, en eksperimenterende system ved hjælp af FRAP for at evaluere zygotisk kromatin løshed blev udviklet. Fordi dette var en ny observationssystem til zygotes, var det kaldes zygotisk FRAP (zFRAP). zFRAP påvirkede ikke kritisk fuldbårne udvikling og har været rapporteret et andet sted7. I denne betænkning, er protokollen af denne eksperimentelle metode beskrevet.

<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

Live imaging er et kraftfuldt værktøj, der giver mulighed for analyse af molekylære begivenheder under ontogenesis. For nylig, kromatin løshed eller åbenhed har vist sig at være involveret i cellulære differentiering potentiale af pluripotente stamceller fra embryoner. Det blev tidligere rapporteret, sammenlignet med embryonale stamceller, zygotes havnen en ekstremt løsnede kromatin struktur, tyder på sin forening med deres totipotency. Men indtil nu, det er ikke blevet behandlet om denne yderst løsnes/open kromatin struktur er vigtigt for embryonale udviklingsmæssige potentiale. I den foreliggende undersøgelse, for at undersøge denne hypotese, blev en eksperimentel system i hvilke zygotes, der blev analyseret af fluorescens opsving efter foto-blegning kan udvikle sigt uden nogen betydelige skader udviklet. Vigtigst, systembehov denne eksperimentelle kun Termokande-plade varmelegeme ud over en Konfokal laser scanning mikroskop. Resultaterne af denne undersøgelse tyder at fluorescens opsving efter foto-blegning analyse (FRAP) analyse kan bruges til at undersøge, om de molekylære begivenheder i zygotisk kromatin vigtigt for fuldbårne udvikling.

zFRAP analyse med eGFP-H2B
Ordentligt produceret blev mRNA kodning eGFP-H2B, som ses som en skiftede bandet forårsaget af poly en hale (figur 2A), sprøjtet ind i cytoplasma af zygotes med en 2. polar kroppen på 1-3 h efter insemination (figur 2B). Otte til 12 h efter insemination, zygotes med 2 pronuclei viser udtryk for eGFP-H2B blev indsamlet og zFRAP analyseret (<str...

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 løshed synes at være involveret i den udviklingsmæssige potentiale af blastomeres. Det vides dog ikke, om kromatin løshed kan bruges som en pålidelig indeks for den udviklingsmæssige potentiale for embryoner. Her, er blevet beskrevet en eksperimentel system hvor kromatin løshed-evalueret zygotes kan udvikle sig til fuld sigt.

Zygotisk fluorescens opsving efter foto-blegning analyse for kromatin løshed, der giver mulighed for fuld-sigt udvikling

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

Måling Protein Stabilitet i Living zebrafisk embryoner anvendelse af fluorescens Decay Efter fotoomdannelse (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

Genom-dækkende Snapshot af Chromatin Regulators og stater i<em&gt; Xenopus</em&gt; Embryoner af 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

Protokoller til Indhentning zygotiske og somatiske for studere regulering af Early Embryo Udviklingen i 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 af Cardiomyocyte Development hjælp Immunofluorescens i embryonale Mouse Hjerte

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 af zebrafisk Larver Skeletmuskel Integritet med Evans blå farvestof

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 og karakterisering af muse Antral Oocyter Baseret på nucleolar 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

Analyse af zebrafisk Kidney Udvikling med Time-lapse Imaging Ved hjælp af en dissektionsmikroskop Udstyret til Optical Sektionsinddeling

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

Brug Light Sheet Fluorescens mikroskopi til billede zebrafisk 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

Udvikling af et<em&gt; In vitro</em&gt; Assay til Evaluer kontraktionsfunktion af mesenchvmal-celler der undergik Epithelial-Mesenchymal Overgangseffekter

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

Beregningsmæssige analyse af Caenorhabditis elegans kønscelleoverførsel at studere Distribution kerner, proteiner og cytoskelettet

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