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.

NOTE: Toutes les expériences ont été approuvées par le soin et l&#39;utilisation des animaux Commission institutionnelle à l&#39;Université de Caroline du Nord à Chapel Hill. 1. accise de la valve aortique <ol><li> En utilisant une technique d&#39;euthanasie approuvé pour le stade embryonnaire d&#39;intérêt, euthanasier une souris enceinte de chiots à la journée embryonnaire souhaitée (E) de développement. </li><li> Utiliser 70% d&#39;éthanol pour stériliser l&#39;abdomen de la femme enceinte. Soulevez la peau et les muscles du bas-ventre et à l&#39;écart des organes internes, et de disséquer cavité abdominale inférieure ouverte la femme de permettre l&#39;accès à la corne utérine. </li><li> Pour récupérer la corne utérine, maintenez le col avec une pince et caudale soigneusement découpées à la pince. Soulevez la corne utérine, la coupe de tout le tissu conjonctif qui maintient la corne en place, et terminer la suppression en coupant la jonction de la corne de l&#39;utérus avec l&#39;oviducte. </li><li> Placez la corne utérine disséqué dans un col boîte de Pétri contenantd 0,1 M de tampon Tris (pH 7,6) et rincer au besoin. Ensuite, couper la corne utérine ouverte sens de la longueur pour exposer les sacs embryonnaires. </li><li> Pour exposer l&#39;embryon, couper ouvrir un sac embryonnaire à la jonction du placenta et de l&#39;embryon et couper les vaisseaux ombilicaux pour libérer l&#39;embryon. Placez l&#39;embryon disséqué dans une deuxième boîte de Pétri avec un tampon froid Tris 0,1 M. Retour de la première boîte de Pétri avec les embryons restants undissected à glace jusqu&#39;au moment de la prochaine embryon. </li><li> Pour améliorer l&#39;accès de la paroi thoracique dans l&#39;embryon, décapiter l&#39;embryon. Si le génotypage est nécessaire, enlever et sauver un morceau de tissu supplémentaire dans un tube Eppendorf placé sur la glace. </li><li> Placer l&#39;embryon sur le dos, et couper la paroi thoracique à la verticale le long du côté de la cage thoracique, à proximité d&#39;un membre antérieur, et horizontalement au-dessus du diaphragme pour visualiser le coeur. Ensuite, ouvrez la paroi thoracique à exposer le cœur. </li><li> En utilisant une pince pour tenir haut le long des gros vaisseaux, soulever le cœur à l&#39;aide de pinces et couper les navires de moins. Lan, couper au-dessus de la pince pour libérer le cœur. Si les vaisseaux pulmonaires restent non coupés, les poumons peuvent être enlevées avec le cœur et doivent être retirés avant de poursuivre. </li><li> Couper et enlever l&#39;artère pulmonaire au-dessus du niveau de la vanne; aux stades embryonnaires décrits ici, l&#39;artère pulmonaire est légèrement opaque, ce qui facilite sa discrimination de la région de la soupape. Coupez juste en dessous de la valve pulmonaire, en évitant le myocarde ventriculaire trabéculaire et de recueillir comme décrit à l&#39;étape 1.10 si cette région est également intéressant. Ici et dans l&#39;étape suivante, utilisez l&#39;action capillaire pour attirer tampon Tris excès loin de la zone d&#39;intérêt avant de recueillir dans un tampon de lyse. </li><li> Retirer soigneusement la aorte distale de la valvule aortique, juste au-dessus du niveau de la vanne; comme l&#39;artère pulmonaire, l&#39;aorte reste légèrement opaque à ces stades embryonnaires, ce qui facilite son extraction. Coupez juste en dessous de la valve aortique, en évitant encore le myocarde ventriculaire trabéculaire, et transférer levanne à l&#39;aide de pinces dans un tube Eppendorf avec du tampon de lyse 2 ul (décrit dans le tableau des matériaux) contenant des inhibiteurs de protease et de phosphatase sur de la glace. </li><li> Répétez les étapes 1.6 à 1.10 jusqu&#39;à ce que les vannes ont été excisés de tous les embryons, la collecte de chaque valve dans un tube Eppendorf séparé. Si tous les embryons ont le même génotype, toutes les vannes peuvent être collectées dans un seul tube Eppendorf. </li><li> Si génotypage doit être effectuée afin de déterminer les vannes à mettre en commun, mettre immédiatement les échantillons dans un tampon de lyse à -80 ° C jusqu&#39;à utilisation ultérieure. Les échantillons peuvent ensuite être regroupées après génotypage (étape 2.1.1). </li></ol> Extraction de la protéine 2 <ol><li> Décongeler les tissus prélevés sur la glace et centrifuger à 13 100 g pendant 1 min pour recueillir les tissus dans un tampon de lyse au fond du tube. <ol><li> Si les embryons ont été génotypés, utiliser une pipette pour recueillir doucement et le tampon de lyse piscine contenant les régions de soupape à partir d&#39;embryons avec des génotypes similaires. Pour assurer une forte bet, la mise en commun des régions 3-4 de soupape à partir d&#39;embryons E13.5 par échantillon est recommandé. </li><li> Si tous les embryons sont du même génotype et toutes les régions de la vanne ont été rassemblées à l&#39;étape 1.10, lyser l&#39;ensemble de l&#39;échantillon, comme décrit dans l&#39;étape 2.2. </li></ol></li><li> Vérifiez le volume de l&#39;échantillon global résultant et ajouter un tampon de lyse à un volume total de 40 ul; alors, perturber et homogénéiser les échantillons à l&#39;aide de 5 mm billes d&#39;acier inoxydable dans des tubes Eppendorf. Actionnez le lyser pour 2-4 min à 50 Hz, selon les instructions du fabricant. tubes à centrifuger brièvement, (~ 13 100 x g pendant 1 min), et de transfert des échantillons dans de nouveaux tubes, laissant derrière les débris insolubles. </li><li> Préparer des échantillons distincts pour SDS-PAGE et transfert ultérieur pour analyse western blot, à la suite d&#39;un protocole tel que Eslami et al 9. Buvard pour une protéine de contrôle du chargement est essentiel pour la quantification des protéines. </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. 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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. 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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 capacité à quantifier les niveaux de protéines au début embryon de souris et poussin régions cardiaques de soupape constitue un outil supplémentaire pour comprendre les événements de signalisation cellulaires critiques pour le développement de la vanne. Notre protocole décrit ici ne diffère pas beaucoup de procédures d&#39;isolement de protéines standard. Cependant, en modifiant quelques étapes clés, nous avons réussi à obtenir des protéines phosphorylées de tailles très petits échantillons. Pour...

Etre capable d&#39;identifier et de quantifier les niveaux d&#39;expression de protéine est une technique standard pour animale et des expériences basées sur des cellules. Cependant, malgré un intérêt de longue date dans le développement de valve cardiaque embryonnaire précoce, l&#39;évaluation de l&#39;expression des protéines dans ce tissu spécifique au cours du développement est actuellement limité à l&#39;immunohistochimie à la fois le poussin et 1,2 de la souris. Une partie de la difficulté à quantifier l&#39;expression des protéines dans les soupapes de développement de la plupart des organismes modèles (par exemple, poulet et de souris) est la petite taille des vannes, ce qui limite la quantité de protéine qui peut être obtenue. Ainsi, pour des analyses quantitatives, les chercheurs s&#39;appuient généralement sur ​​l&#39;extraction de l&#39;ARN et l&#39;amplification de la PCR quantitative ultérieure ou l&#39;analyse des microréseaux 2-5. Cependant, les niveaux d&#39;expression d&#39;ARN et de protéines ne sont pas entièrement corrélative 6, donc en mettant l&#39;accent sur ​​l&#39;expression de l&#39;ARN ne peuvent pas fournir un compte rendu rigoureux des nombreux changements qui se produisent dans toute signalisation donnévoie à différents moments de l&#39;évolution. Sur la base de cette limite dans la méthodologie actuellement disponibles, l&#39;objectif de cette procédure était de développer un protocole pour obtenir de manière fiable des quantités suffisantes de protéine à partir de la souris embryonnaire régions de valve cardiaque en développement pour l&#39;analyse quantitative des changements qui se produisent dans différentes voies de signalisation qui sont importantes dans la maturation de ce tissu. Vannes embryonnaires sont déjà couramment disséqués de souris pour l&#39;isolement de l&#39;ARN et l&#39;analyse ultérieure gène d&#39;expression 2-5. Toutefois, ces études ont été limitées à l&#39;utilisation de l&#39;expression des gènes en tant que sortie de lecture de signalisation activation de la voie, ce qui ne permet pas la détection de détecter des modifications post-traductionnelles des protéines qui sont susceptibles d&#39;affecter la signalisation en aval. En utilisant les techniques d&#39;isolation de l&#39;ARN comme un point de départ, nous avons commencé par disséquer les régions d&#39;intérêt. Parce que notre intérêt détectait protéines phosphorylées qui sont des indices de pat signalisationHway activité pendant une période spécifique du développement de la valve aortique (E13.5-14.5), on a effectué toutes les dissections dans du tampon Tris-phosphate libre et recueilli les soupapes dans un tampon de lyse à base de Tris avec des inhibiteurs de phosphatase et protéase. Dans notre cas particulier, que les régions de la valve aortique ont été recueillies, mais la région de la valve pulmonaire peuvent facilement être obtenues en même temps. Les régions de soupapes sont ensuite homogénéisé et combiné avec un tampon d&#39;échantillon qui est actuellement utilisé pour étudier l&#39;expression des protéines dans les valves cardiaques adultes 7. En utilisant des volumes d&#39;échantillon de petite taille (par exemple, 2 pi) et la mise en commun des régions de soupape à partir d&#39;embryons avec le même génotype, nous avons pu détecter des protéines phosphorylées et non phosphorylés à jour embryonnaire 13,5 8. Étant donné que les régions de la vanne peuvent être congelés et stockés dans un tampon de lyse, les embryons peuvent être génotypés si nécessaire avant la mise en commun. Cette technique élargit l&#39;ensemble des outils qui sont disponibles pour l&#39;évaluation signalin cellulaireg voies au cours du développement et fournit un compliment quantitative de l&#39;immunohistochimie, en particulier pour les valves cardiaques en développement. Cette technique devrait être bénéfique non seulement pour les cardiologues de développement, mais aussi à tous les biologistes du développement qui travaillent avec des embryons de stade précoce sont intéressés dans les régions qui contiennent des tissus limitée.

<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.

Grâce à cette technique de préparation, nous avons pu détecter phosphorylée Smad 1,5,8 (pSmad) dans les régions de la valve aortique unique à partir de E13.5 embryons. Comme le montre la Figure 1A, la protéine isolée à partir d&#39;une même région de la soupape est suffisant pour détecter une bande de pSmad faible. L&#39;intensité du signal augmente proportionnellement au nombre de régions de soupapes qui sont mises en commun. Fait important, la pSmad rapport a / β-actine rest...

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.

Protein Isolation du développement embryonnaire Souris Coeur Région Valve

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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9.3K vues.  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.

Vidéo: 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-dissection du cerveau de rat pour l&#39;ARN ou de l&#39;extraction de protéines à partir région spécifique du cerveau

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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Biologie

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;isolement et la dérivation de cellules germinales embryonnaires de souris

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.

Isolement et culture de cellules de la zone néphrogénique du rein d&#39;embryon de souris

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.

Immunocoloration des Disséqué coeur embryonnaire Zebrafish

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.

Génération de souris dérivées de cellules souches pluripotentes induites

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;

Isoler des cellules mélanocytes comme primaires du Cœur de souris

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; Méthode de la culture pour le embryonnaire Souris Coeur

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.

Isolement des cellules endothéliales embryonnaires ventriculaires

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.

Cellule étiquetage et l&#39;injection dans le développement embryonnaires coeurs de souris

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.

Isolement des cellules endothéliales murines Valve

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