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

Ringraziamo Satoshi Kishigami, Sayaka Wakayama, Hiroaki Nagatomo, Satoshi Kamimura e Kana Kishida per fornire supporto tecnico e commenti critici. Questo lavoro è stato parzialmente finanziato dal Ministero della pubblica istruzione, cultura, sport, scienza e tecnologia programma per promuovere la riforma delle università nazionali al modus operandi; la società giapponese per la promozione della scienza (16h 02593), Asada Science Foundation e la Fondazione di scienza di Takeda a T.W. I finanziatori non avevano alcun ruolo nel disegno dello studio, raccolta dati e analisi, decisione di pubblicare o preparazione del manoscritto.

Questo studio è stato svolto in stretta conformità con le raccomandazioni della Guida per la cura e l'uso di animali da laboratorio dell'Università di Yamanashi. Il protocollo è stato approvato dal comitato etico di esperimenti di animale dell'Università di Yamanashi (permesso numero: A24-50). Tutti gli interventi sono stati eseguiti in anestesia tribromoethanol, e tutti gli sforzi sono stati fatti per ridurre al minimo la sofferenza. Una panoramica illustrata delle procedure e l'orario sono mostrati in <strong clas...

<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>

Come rivelato in questo studio, l'analisi di zFRAP non causa danni critici al termine sviluppo, suggerendo che questo metodo è uno strumento molto utile per rivelare l'associazione tra eventi molecolari e il potenziale dello sviluppo embrionale. Durante la riprogrammazione, in due-cellula come stato di cellule ES, da cui il potenziale differenziale di quelle cellule diventa alto quanto quello di embrioni in fase bicellulare, scioltezza di cromatina cambia in quello paragonabile con fase bicellulare embrioni<sup class="x...

Dopo la fecondazione, la struttura della cromatina è alterata in modo dinamico e struttura della cromatina zygotic è poi alla fine stabilito1,2. Durante questo periodo, nel paterni pronuclei, le proteine della cromatina dominante sono trasformata da protamina istone. La cromatina risultante è estremamente diversa da quella di spermatozoi e ovociti femminili in diversi punti (ad es., composizione variante dell'istone, modifica dell'istone). Così, formato della cromatina zygotic è pensata per essere importante per il successivo sviluppo embrionale. Tuttavia, nonostante gli sforzi per rivelare i dettagli della struttura della cromatina zygotic per lunghi periodi, non sono mai stati metodi per valutare la qualità degli zigoti o per predire il loro sviluppo di termine nella fase di un cella analizzando la loro struttura della cromatina stabilito.
Nello studio precedente, si è scoperto che zigoti hanno una struttura della cromatina estremamente allentata3. Attualmente, scioltezza della cromatina o apertura è creduta per essere un fattore importante per la differenziazione cellulare potenziale di cellule staminali embrionali (ES)4. Le cellule ES non esibiscono omogeneità nella natura, ma sono piuttosto eterogenee; nelle colonie di cellule ES, alcuni transitoriamente acquisire un maggiore potenziale di differenziazione paragonabile a blastomeri degli embrioni in fase due-cellula. Durante questa transizione in due-cellula come stato, scioltezza della cromatina in ES cellule si trasforma in che cosa è paragonabile con fase bicellulare embrioni5. Così, scioltezza di cromatina sembra essere importante per potenziale di differenziazione cellulare ed è possibile che ampiamente aperto della cromatina in zigoti è utile per la valutazione del potenziale inerente allo sviluppo zygotic.
La formazione immagine dal vivo è un potente strumento che permette l'analisi di eventi molecolari durante l'ontogenesi in quanto questo metodo permette lo sviluppo successivo e anche pieno-termine sviluppo6. Come uno dei metodi di formazione immagine diretta, FRAP analisi sono stato utilizzato per esaminare la scioltezza della cromatina in embrioni preimpianto ed ES cellule3,4,5. Se scioltezza zygotic cromatina possa essere analizzato senza un effetto negativo sullo sviluppo di pieno-termine di FRAP analisi, può essere un valido strumento per la valutazione della qualità degli embrioni nella fase uno-cella. Tuttavia, non sono stati esaminati gli effetti sullo sviluppo di pieno-termine da questo metodo sperimentale. Recentemente, è stato sviluppato un sistema sperimentale utilizzando FRAP per valutare la scioltezza della cromatina zygotic. Perché questo era un nuovo sistema di osservazione per zigoti, esso è stato chiamato come zygotic FRAP (zFRAP). zFRAP non ha colpito criticamente termine sviluppo è stato segnalato altrove7. In questo rapporto, il protocollo di questo metodo sperimentale è descritto.

<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

La formazione immagine dal vivo è un potente strumento che permette l'analisi di eventi molecolari durante l'ontogenesi. Recentemente, la scioltezza della cromatina o apertura ha dimostrato di essere coinvolti nel potenziale differenziazione cellulare delle cellule staminali embrionali pluripotenti. Precedentemente è stato segnalato che confrontato con cellule staminali embrionali, zigoti harbor una struttura della cromatina estremamente allentata, suggerendo la sua associazione con la loro totipotenza. Tuttavia, fino ad ora, è non stato affrontato sia la struttura della cromatina estremamente allentata/Apri, è importante per il potenziale dello sviluppo embrionale. Nello studio presente, per esaminare questa ipotesi, è stato sviluppato un sistema sperimentale in cui zigoti che sono stati analizzati tramite fluorescenza recupero dopo lo sbiancamento foto può sviluppare a termine senza alcun danno significativo. D'importanza, questo sistema sperimentale deve solo thermos-piastra di riscaldamento oltre a un microscopio a scansione confocale del laser. I risultati di questo studio suggeriscono che il recupero fluorescenza dopo lo sbiancamento foto analisi (FRAP) analisi può essere utilizzato per studiare se gli eventi molecolari nella cromatina zygotic sono importanti per lo sviluppo di full-term.

zFRAP analisi con eGFP-H2B
Prodotto correttamente mRNA codificante eGFP-H2B, che è visto come una band spostata causato da poli una coda (Figura 2A), è stato iniettato nel citoplasma degli zigoti con un 2 ° corpo polare a 1-3 h dopo l'inseminazione (Figura 2B). Otto a 12 h dopo l'inseminazione, zigoti con 2 pronuclei mostrando l'espressione di eGFP-H2B sono stati raccol...

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

Scioltezza di cromatina sembra essere coinvolto nel potenziale inerente allo sviluppo dei blastomeri. Tuttavia, non è noto se scioltezza della cromatina può essere utilizzato come un indice affidabile per il potenziale di sviluppo per gli embrioni. Qui, è stato descritto un sistema sperimentale in cui zigoti scioltezza-valutata della cromatina possono sviluppare al termine completo.

Fluorescenza zygotic recupero dopo lo sbiancamento foto analisi per scioltezza di cromatina che permette lo sviluppo di Full-term

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

Misurare Protein Stabilità in Living embrioni di zebrafish Usando fluorescenza Decay Dopo Fotoconversione (FDAP)

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

Ricerca

Biologia dello sviluppo

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

Genoma Snapshot dei regolatori della cromatina e membri<em&gt; Xenopus</em&gt; Gli embrioni da 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

Protocolli per ottenere Zygotic e Somatic embrioni per lo studio della regolazione dello sviluppo embrionale precoce nel Modello leguminose<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

Analisi di Cardiomyocyte Sviluppo usando immunofluorescenza in cuore embrionali di topo

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

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans 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

Isolamento e caratterizzazione di mouse Antral ovociti Sulla base Nucleolare cromatina Organizzazione

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

Analisi dello sviluppo del rene Zebrafish con time-lapse imaging Utilizzando una dissezione microscopio dotato di sezionamento ottico

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

Utilizzando tecnica LUCE microscopia a fluorescenza all&#39;Immagine sviluppo Zebrafish Eye

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

Sviluppo di un<em&gt; In Vitro</em&gt; Test per valutare funzione contrattile del mesenchimali Le cellule che sono stati sottoposti a transizione epitelio-mesenchimali

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

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

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