We would like to thank Andrea Portbury and Davin Townley-Tilson for critical reading of the manuscript and the NIH (grant # R01HL061656) for funding support.

NOTA: Tutti gli esperimenti sono stati approvati dalla cura e uso degli animali Comitato Istituzionale presso l&#39;Università del North Carolina a Chapel Hill. 1 Excise della valvola aortica <ol><li> Utilizzando una tecnica di eutanasia approvato per la fase embrionale di interesse, eutanasia di un mouse incinta con cuccioli al giorno embrionale desiderata (E) dello sviluppo. </li><li> Utilizzare etanolo al 70% per sterilizzare l&#39;addome della femmina incinta. Sollevare la pelle ei muscoli del basso addome alto e lontano dagli organi interni, e sezionare cavità inferiore addominale open femminile per consentire l&#39;accesso al corno uterino. </li><li> Per recuperare il corno uterino, tenere il collo dell&#39;utero con una pinza e caudale con attenzione tagliare le pinze. Sollevare il corno uterino, tagliando qualsiasi tessuto connettivo che tiene il corno in luogo, e completare la rimozione tagliando la giunzione del corno uterino con l&#39;ovidotto. </li><li> Posizionare il corno uterino sezionato in una capsula di Petri contenente cold 0.1 M tampone Tris (pH 7.6) e sciacquare come necessario. Quindi, tagliare il corno uterino longitudinalmente per esporre i sacchi embrionali aperto. </li><li> Per esporre l&#39;embrione, tagliare aprire un sacco embrionale allo svincolo della placenta e l&#39;embrione e tagliare i vasi ombelicali per liberare l&#39;embrione. Posizionare l&#39;embrione sezionato in un secondo piatto di Petri con freddo 0.1 M tampone Tris. Ritorna il primo piatto di Petri con i rimanenti embrioni undissected per ghiaccio fino pronto per la prossima embrione. </li><li> Per migliorare l&#39;accesso della parete toracica all&#39;interno dell&#39;embrione, decapitare il embrione. Se genotipizzazione è necessario, rimuovere e salvare un pezzo di tessuto in più in una provetta Eppendorf posti su ghiaccio. </li><li> Inserire l&#39;embrione sul dorso, e tagliare la parete toracica verticalmente lungo il lato della gabbia toracica, vicino a una zampa anteriore, e orizzontalmente sopra il diaframma di visualizzare il cuore. Quindi, aprire la parete toracica per esporre il cuore. </li><li> Utilizzando pinze per tenere alto lungo i grossi vasi, sollevare il cuore con pinze e tagliare i vasi di seguito. Iln, tagliare sopra la pinza per liberare il cuore. Se i vasi polmonari rimangono non tagliata, i polmoni possono essere rimossi con il cuore e devono essere rimosse prima di continuare. </li><li> Tagliare e rimuovere l&#39;arteria polmonare di sopra del livello della valvola; nelle fasi embrionali qui descritte, l&#39;arteria polmonare è leggermente opaca, che aiuta nella sua discriminazione dalla regione valvola. Tagliare appena sotto la valvola polmonare, evitando il miocardio ventricolare trabeculated e raccogliere come descritto al punto 1.10, se questa regione è anche di interesse. Qui e nella fase successiva, utilizzare un&#39;azione capillare per disegnare tampone Tris eccesso di distanza dalla regione di interesse prima di raccogliere in tampone di lisi. </li><li> Rimuovere accuratamente il distale dell&#39;aorta alla valvola aortica, appena sopra il livello della valvola; come l&#39;arteria polmonare, l&#39;aorta rimane leggermente opaca in queste fasi embrionali, aiutando nella sua rimozione. Tagliare appena sotto la valvola aortica, ancora una volta evitando il miocardio ventricolare trabeculated, e trasferire ilvalvola con pinze ad una provetta Eppendorf con tampone di lisi 2 microlitri (descritto nella tabella materiali) contenente inibitori di proteasi e fosfatasi sul ghiaccio. </li><li> Ripetere i passaggi 1,6-1,10 finché le valvole sono stati rimossi da tutti gli embrioni, raccogliendo ogni valvola in un tubo Eppendorf separata. Se tutti gli embrioni hanno lo stesso genotipo, tutte le valvole possono essere raccolte in un unico tubo Eppendorf. </li><li> Se genotipizzazione deve essere eseguito per determinare quali valvole di riunire insieme, immediatamente mettere i campioni in tampone di lisi a -80 ° C fino a nuovo uso. I campioni possono quindi essere raggruppati dopo genotipizzazione (punto 2.1.1). </li></ol> Estrazione 2 Proteine <ol><li> Scongelare tessuti raccolti su ghiaccio e centrifugare a 13.100 xg per 1 min per raccogliere i tessuti in un tampone di lisi sul fondo della provetta. <ol><li> Se sono stati genotipizzati embrioni, utilizzare una pipetta per raccogliere delicatamente e piscina di buffer di lisi contenente le regioni valvole da embrioni con genotipi simili. Per garantire un forte be, riunendo 3-4 regioni valvole da embrioni E13.5 per si raccomanda campione. </li><li> Se tutti gli embrioni erano dello stesso genotipo e tutte le regioni valvole sono stati raccolti insieme nel passo 1.10, lisare l&#39;intero campione, come descritto al punto 2.2. </li></ol></li><li> Controllare il volume del campione composito risultante e aggiungere tampone di lisi per un volume totale di 40 microlitri; poi, interrompere e omogeneizzare campioni utilizzando perline in acciaio inox 5 mm provette Eppendorf. Azionare il Lyser per 2-4 min a 50 Hz, come da istruzioni del produttore. Tubi Spin brevemente, (~ 13.100 xg per 1 minuto), e il trasferimento dei campioni ai nuovi tubi, lasciando dietro di sé i detriti insolubile. </li><li> Preparare e campioni separati per SDS-PAGE e trasferimento per la successiva analisi Western Blot, a seguito di un protocollo come Eslami et al 9. Blotting per una proteina di controllo di carico è essenziale per la quantificazione delle proteine. </li></ol>

<ol>
	<li>Wirrig, E. E., Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+cartilage+and+bone-related+proteins+in+pediatric+and+adult+diseased+aortic+valves.">Differential expression of cartilage and bone-related proteins in pediatric and adult diseased aortic valves.</a> J Mol Cell Cardiol. 50, 561-569 (2011).</li><li>Chakraborty, S., 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=Twist1+promotes+heart+valve+cell+proliferation+and+extracellular+matrix+gene+expression+during+development+in+vivo+and+is+expressed+in+human+diseased+aortic+valves.">Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves.</a> Dev Biol. 347, 167-179 (2010).</li><li>Lee, M. P., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twist1+directly+regulates+genes+that+promote+cell+proliferation+and+migration+in+developing+heart+valves.">Twist1 directly regulates genes that promote cell proliferation and migration in developing heart valves.</a> PLoS One. 6 (e29758), (2011).</li><li>Peacock, J. D., Huk, D. J., Ediriweera, H. N., Lincoln, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox9+transcriptionally+represses+Spp1+to+prevent+matrix+mineralization+in+maturing+heart+valves+and+chondrocytes.">Sox9 transcriptionally represses Spp1 to prevent matrix mineralization in maturing heart valves and chondrocytes.</a> PLoS One. 6 (e26769), (2011).</li><li>Chakraborty, S., Cheek, J., Sakthivel, B., Aronow, B. J., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+gene+expression+profiles+in+developing+heart+valves+and+osteoblast+progenitor+cells.">Shared gene expression profiles in developing heart valves and osteoblast progenitor cells.</a> Physiol Genomics. 35, 75-85 (2008).</li><li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nat Rev Genet. 13, 227-232 (2012).</li><li>Meng, X., 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=Expression+of+functional+Toll-like+receptors+2+and+4+in+human+aortic+valve+interstitial+cells:+potential+roles+in+aortic+valve+inflammation+and+stenosis.">Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells: potential roles in aortic valve inflammation and stenosis.</a> Am J Physiol Cell Physiol. 294, 29-35 (2008).</li><li>Dyer, L. A., Wu, Y., Moser, M., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPER-induced+BMP+signaling+promotes+coronary+artery+remodeling.">BMPER-induced BMP signaling promotes coronary artery remodeling.</a> Developmental Biology. 386, 385-394 (2014).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Garside, V. C., Chang, A. C., Karsan, A., Hoodless, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-ordinating+Notch,+BMP,+and+TGF-beta+signaling+during+heart+valve+development.">Co-ordinating Notch, BMP, and TGF-beta signaling during heart valve development.</a> Cell Mol Life Sci. 70, 2899-2917 (2013).</li><li>Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P., Sucov, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+mammalian+cardiac+neural.">Fate of the mammalian cardiac neural.</a> Development. 127, 1607-1616 (2000).</li><li>Scherptong, R. W., 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=Morphogenesis+of+outflow+tract+rotation+during+cardiac+development:+the+pulmonary+push+concept.">Morphogenesis of outflow tract rotation during cardiac development: the pulmonary push concept.</a> Dev Dyn. 241, 1413-1422 (2012).</li><li>Mohler, E. R., Adam 3rd, L. P., McClelland, P., Graham, L., Hathaway, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+osteopontin+in+calcified+human+aortic+valves.">Detection of osteopontin in calcified human aortic valves.</a> Arterioscler Thromb Vasc Biol. 17, 547-552 (1997).</li></ol>

The authors have nothing to disclose.

La capacità di quantificare i livelli di proteine ​​nei primi mesi del topo embrionale e pulcino regioni della valvola cardiaca fornisce un ulteriore strumento per la comprensione degli eventi di segnalazione cellulari critici per lo sviluppo della valvola. Il nostro protocollo qui descritto non differisce molto da procedure di isolamento delle proteine ​​standard. Tuttavia, modificando alcuni passaggi chiave, abbiamo ottenuto con successo proteine ​​fosforilate da estremamente campioni di piccole dimension...

Essere in grado di identificare e quantificare i livelli di espressione della proteina è una tecnica standard per ragioni sanitarie e di esperimenti basati su celle. Tuttavia, nonostante una lunga interesse per lo sviluppo della valvola cardiaca embrionale precoce, valutando l&#39;espressione della proteina in questo tessuto specifico durante lo sviluppo è attualmente limitato a immunoistochimica sia il pulcino e il mouse 1,2. Parte della difficoltà di quantificare l&#39;espressione della proteina nelle valvole di sviluppo della maggior parte degli organismi modello (ad esempio, ceci e topo) è la piccola dimensione delle valvole, che limita la quantità di proteine ​​che si possono ottenere. Così, per analisi quantitative, i ricercatori in genere si basano su l&#39;estrazione dell&#39;RNA e l&#39;amplificazione per la successiva PCR quantitativa o analisi microarray 2-5. Tuttavia, i livelli di espressione di RNA e proteine ​​non sono del tutto correlativo 6, in modo da concentrarsi su espressione di RNA non è in grado di fornire una rigorosa conto dei numerosi cambiamenti che si verificano in una determinata segnalazionepercorso in diversi momenti durante l&#39;evoluzione. Sulla base di questo limite nella metodologia attualmente disponibili, l&#39;obiettivo di questa procedura è stato quello di sviluppare un protocollo per ottenere in modo affidabile una quantità sufficiente di proteine ​​dalle embrionali di topo in via di sviluppo le regioni valvola cardiaca per l&#39;analisi quantitativa dei cambiamenti che si verificano in diverse vie di segnalazione che sono importanti in la maturazione di questo tessuto. Le valvole embrionali sono già comunemente dissezionati da topi per l&#39;isolamento di RNA e la successiva analisi di espressione genica 2-5. Tuttavia, questi studi si sono limitati a usare l&#39;espressione genica come out lettura di segnalare l&#39;attivazione della via, che non consente il rilevamento di rilevare modificazioni post-traduzionali di proteine ​​che possono influenzare la segnalazione a valle. Utilizzando le tecniche di isolamento dell&#39;RNA come punto di partenza, abbiamo iniziato con dissezione delle regioni di interesse. Perché il nostro interesse è stato rilevando proteine ​​fosforilate che erano indicativi di pat segnalazioneattività HWAY durante un periodo specifico di sviluppo della valvola aortica (E13.5-14.5), abbiamo effettuato tutte le dissezioni in tampone Tris senza fosfati e raccolto le valvole in un tampone di lisi a base di Tris con inibitori della proteasi e fosfatasi. Nel nostro caso specifico, solo le regioni valvola aortica sono stati raccolti, ma la regione valvola polmonare possono essere facilmente ottenuti allo stesso tempo. Le regioni valvole erano poi omogeneizzato e combinato con un tampone che viene attualmente utilizzato per studiare l&#39;espressione della proteina in valvole cardiache adulte 7. Utilizzando piccoli volumi di campione (ad esempio, 2 microlitri) e la messa in comune delle regioni valvole da embrioni con lo stesso genotipo, siamo stati in grado di rilevare le proteine ​​fosforilate e nonphosphorylated al giorno embrionale 13.5 8. Poiché le regioni di valvole possono essere congelati e conservati in tampone di lisi, gli embrioni possono essere genotipizzati se necessario prima di pooling. Questa tecnica amplia il set di strumenti che sono disponibili per la valutazione signalin cellulareg percorsi durante lo sviluppo e fornisce un complimento quantitativo di immunoistochimica, specificatamente per le valvole cardiache in via di sviluppo. Questa tecnica dovrebbe essere di beneficio non solo ai cardiologi di sviluppo, ma anche a tutti i biologi dello sviluppo che lavorano con gli embrioni in fase iniziale sono interessati nelle regioni che contengono tessuti limitata.

<table border="0" cellpadding="0" cellspacing="0" width="972">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:512px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Timed-pregnant mouse</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>To be dissected at the embryonic stage of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereoscopic microscope</td>
			<td style="width:125px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1 M Tris, pH 7.6</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscissors</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fine forceps, #5</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:119px;">11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting needle holders</td>
			<td style="width:125px;">Ted Pella Inc.</td>
			<td style="width:119px;">13560</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting needles</td>
			<td style="width:125px;">Ted Pella Inc.</td>
			<td style="width:119px;">13561-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micropipette, 20 &#956;l, with tips</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysis buffer</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PhosSTOP</td>
			<td style="width:125px;">Roche</td>
			<td style="width:119px;">4906845001</td>
			<td>Add 1 tablet to 10 ml lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TissueLyser LT</td>
			<td style="width:125px;">Qiagen</td>
			<td style="width:119px;">85600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel beads</td>
			<td style="width:125px;">Qiagen</td>
			<td align="right">69989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protein isolation from the developing embryonic mouse heart valve region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

Utilizzando questa tecnica di preparazione, siamo stati in grado di rilevare fosforilata Smad 1,5,8 (pSmad) nelle singole regioni della valvola aortica da E13.5 embrioni. Come mostrato nella Figura 1A, la proteina isolata da anche una sola regione valvola è sufficiente per rilevare una banda pSmad debole. Intensità del segnale aumenta in proporzione al numero di regioni valvole che vengono pool. È importante sottolineare che il / rapporto β-actina pSmad rimane pressoché costante tra le d...

Watch this Scientific Journal Video about Protein Isolation from the Developing Embryonic Mouse Heart Valve Region at JoVE.com

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Proteine ​​Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

protein-isolation-from-developing-embryonic-mouse-heart-valve

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Biology

9.3K Views.  University of North Carolina at Chapel Hill. The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Video: Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissezione del cervello di ratto per estrazione RNA o proteine ​​da Regione specifiche del cervello

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

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Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e coltura di cellule dalla Zona nefrogenica del Rene embrionali di topo

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Immunocolorazione di Dissected Cuore Zebrafish embrionali

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Isolamento di cellule melanociti-come primario dal Cuore del mouse

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolamento della embrionali ventricolari cellule endoteliali

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Etichettatura cellulare e iniezione nello sviluppo embrionali Cuori mouse

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolamento di cellule murine endoteliali Valve

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Proteine ​​Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

Proteine Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione