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.

NOT: Tüm deneyler Chapel Hill&#39;deki North Carolina Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1. Tüketim Aort Kapağı <ol><li> Ilgi embriyonik aşaması için onaylanmış bir ötenazi tekniği kullanılarak, arzu edilen gelişme embriyonik gün (E) yavrular ile hamile bir fareyi euthanize. </li><li> Hamile kadın karnı sterilize etmek için% 70 etanol kullanın. Uzak iç organlardan alt karın yukarı ve cilt ve kas kaldırın ve uterin boynuz erişime izin vermek için açık dişinin alt karın boşluğuna teşrih. </li><li> Uterin boynuzu almak için, forseps forseps ile serviks ve dikkatlice kesilmiş kaudal tutun. Yerinde boynuz tutan herhangi bir bağ dokusu kesme, uterin boynuz kaldırın ve fallop ile uterin horn kavşak keserek kaldırma işlemini tamamlamak. </li><li> Bir Petri kabı içeren col disseke uterin boynuz yerleştirmekd 0.1 M Tris tamponu (pH 7.6) ve gerektiği gibi yıkayın. Sonra, açık uzunluğu-bilge embriyonik keselerinin maruz uterin boynuzu kesti. </li><li> Embriyo göstermek için, plasenta ve embriyo kavşağında bir embriyonik kese kesip ve embriyo serbest göbek damarları kesilmiş. Soğuk 0.1 M Tris tampon içeren ikinci bir Petri kabındaki kesilen embriyo yerleştirin. Sonraki embriyo için hazır olana kadar buz kalan undissected embriyolar ile ilk Petri dönün. </li><li> Embriyonun içinde göğüs duvarı erişimi geliştirmek için, embriyoyu başını kesmek. Genotipleme gerekli ise, kaldırma ve buz üzerine yerleştirilir, bir Eppendorf tüpü içinde ilave bir doku parçası kaydedin. </li><li> Sırtında embriyo yerleştirin ve ön ayakları yakınında, göğüs kafesinin yan boyunca dikey göğüs duvarı kesti ve yatay diyafram üstü kalp görselleştirmek için. Ardından, kalp maruz göğüs duvarı açın. </li><li> Büyük damarların boyunca yüksek tutmak için forseps kullanarak, forseps kullanarak kalp kaldırın ve aşağıdaki damarları kesilmiş.n kalbi boşaltmak için forseps yukarıdaki kesti. Akciğer damarlarında kesilmemiş kalırsa, akciğerler kalp ile kaldırılmış olabilir ve devam etmeden önce çıkarılmalıdır. </li><li> Kesme ve valfın seviyesinden pulmoner arter çıkarmak; Burada tarif edilen embriyonik aşamada, pulmoner arter kapak bölgesinden, ayrımda yardım eden, hafif opaktır. Sadece pulmoner kapak altına indi trabeküler ventriküler miyokard kaçınarak ve bu bölge de ilgi ise adımda 1.10 açıklandığı gibi toplamak. Burada ve bir sonraki aşamada önce, liziz tamponu içinde toplama uzak ilgilenilen bölgesinde aşırı Tris tamponu çizmek için kılcal hareketi vardır. </li><li> Dikkatle vananın seviyesinden, aort kapak aort distalinden kaldırmak; pulmoner arter gibi, aort, uzaklaştırılması yardım, bu embriyonik aşamada biraz opak kalır. Tekrar trabeküle ventriküler miyokard kaçınarak, sadece aort kapak altında kesilmiş ve aktarmakBuz üzerinde proteaz ve fosfataz inhibitörlerini ihtiva eden (Malzeme tabloda tarif edilmiştir) 2 ul liziz tamponu ile bir Eppendorf tüpüne, forseps kullanılarak valf. </li><li> Vanalar ayrı Eppendorf tüp her bir valfi toplayarak, tüm embriyolardan kesilip edilene kadar tekrarlayın 1,6-1,10 adımları. Tüm embriyolar aynı genotipi varsa, tüm supap tek bir Eppendorf tüpü içinde toplanabilir. </li><li> Genotipleme birlikte havuz valflarına belirlemek için gerçekleştirilecekse, hemen bir sonraki kullanıma kadar -80 ° C&#39;de lisis tamponu içinde örnekleri yer. Numuneler daha sonra (adım 2.1.1) genotipleme sonra toplanmış olabilir. </li></ol> 2. Protein Ekstraksiyonu <ol><li> 1 dakika tüpün altındaki liziz tamponunda dokuları toplamak için 13.100 x g buz ve santrifüj üzerine toplanır dokuları çözülme. <ol><li> Embriyolar genotiplendirilmiştir, hafifçe toplamak ve havuz lizis tamponu benzer genotipler ile embriyolar valf bölgeleri içeren bir pipet kullanın. Güçlü bir b sağlamakörnek önerilir başına, E 13.5 embriyolardan 3-4 kapak bölgeleri birleştirilmelidir. </li><li> Tüm embriyolar aynı genotipi ve tüm valf bölgeler Aşama 1,10 araya getirilerek durumunda Adım 2.2 &#39;de tarif edildiği gibi, örneğin tamamına lize. </li></ol></li><li> Elde edilen birleştirilmiş numune miktarını kontrol edin ve 40 ul bir toplam hacim liziz tampon eklemek; Daha sonra, bozabilir ve Eppendorf tüpleri 5 mm paslanmaz çelik boncuklar kullanarak örnekleri homojenize. Üreticinin talimatlarına uygun olarak, 50 Hz&#39;de 2-4 dakika için lize edici çalıştırın. Kısaca Spin tüpler, çözünmeyen enkaz geride bırakarak yeni tüplere (~ 13.100 1 dakika xg), ve transfer örnekleri. </li><li> Hazırlama ve bu Eslami ve ark 9 gibi bir protokol izlenerek, SDS-PAGE ve Western blot analizi daha sonra aktarım için ayrı örnekler. Bir yükleme kontrol proteini için Kurutma protein miktar tayini için önemlidir. </li></ol>

<ol>
	<li>Wirrig, E. E., Hinton, R. B., Yutzey, K. 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The authors have nothing to disclose.

Kalp kapak bölgeleri erken embriyonik fare protein seviyelerini ölçmek ve civciv yeteneği vana gelişimi için kritik hücresel sinyal olayları anlamak için ek bir araç sağlar. Burada tarif Bizim protokol standart protein izolasyon prosedürlerinin büyük ölçüde farklı değildir. Ancak, bazı önemli adımları değiştirerek, biz başarıyla derece küçük örneklem büyüklükleri fosforile proteinleri almış. Bu sonucu elde etmek için, aşağıdaki aşamalar, özel bir önem taşımaktadır. Bu kalite...

Protein ekspresyon seviyelerini belirlemek ve ölçmek için güçlü olmak bir hayvan ve hücre tabanlı deneyler için standart bir tekniktir. Ancak rağmen uzun süreli bir anda civciv ve fare hem de 1,2 immünohistokimya ile sınırlıdır gelişimi sırasında, bu özel doku içindeki protein ifadesi değerlendirilmesi, erken dönemde embriyo gelişimi kalp kapağı ilgi. En model organizmaların (örneğin, civciv ve fare) gelişmekte vanaları protein ifadesini nicel zorluk parçası elde edilebilir protein miktarını sınırlayan vanalar, küçük boyutu. Böylece, kantitatif analiz için, araştırmacılar genellikle müteakip kantitatif PCR veya mikroarray analizi 2-5 RNA ekstraksiyon ve amplifikasyon güveniyor. Bununla birlikte, RNA ve protein ekspresyon seviyeleri 6 tamamen bağıntılı değildir, bu nedenle RNA ekspresyonu odaklanan herhangi bir sinyal meydana gelen pek çok değişiklik sıkı bir hesap sağlayamazgelişmeler sırasında çeşitli zamanlarda yol. Mevcut metodolojisinde bu sınırı dayanarak, bu prosedürün amacı güvenilir önemli olan çeşitli sinyal yollarında meydana gelen değişikliklere kantitatif analiz için gelişmekte embriyonik fare kalp kapak bölgelerden proteinin yeterli miktarda elde etmek için bir protokol geliştirmek oldu Bu doku olgunlaşması. Embriyonik vanalar zaten yaygın RNA izolasyonu ve sonraki gen ekspresyon analizi 2-5 için fareden disseke edilir. Ancak bu çalışmalar, akış aşağı sinyali etkileyebilir translasyon sonrası protein modifikasyonları yer tespit algılanmasını izin vermez yolu aktivasyonunun, bir sinyalleme okuma olarak gen ekspresyonu ile sınırlı kalmıştır. Bir başlangıç ​​noktası olarak RNA izolasyon teknikleri kullanarak, ilgi konusu bölgelerin kesme ile başladı. Bizim faiz fosforillenmiş proteinleri tespit Çünkü sinyalizasyon pat göstergesidir olduğunuaort kapak geliştirme (E13.5-14.5) belirli bir süre boyunca Hway aktivitesi, bu fosfat içermeyen Tris tamponu içinde bütün tahlil yapıldı ve fosfataz ve proteaz inhibitörleri ile bir Tris-bazlı liziz tamponunda valfleri toplanır. Bizim özel durumda, sadece aort kapak bölgeleri toplandı, ancak pulmoner kapak bölgesi kolaylıkla aynı anda elde edilebilir. Valf bölgeleri, homojenize edilir ve şu anda yetişkin kalp valf 7 protein ifadesini incelemek için kullanılan bir numune tampon maddesi ile birleştirildi. Küçük örnek hacimleri (örneğin, 2 ul) kullanılarak ve aynı genotipi ile embriyolardan valf bölgeleri bir araya getirerek, biz embriyonik günde 13.5 8 de fosforlu ve fosforillenmemiş proteinleri tespit başardık. Valf bölgeler dondurulmuş ve lizis tamponunda saklanabilir için araya önce gerekirse, embriyolar genotiplenmiştir edilebilir. Bu teknik, hücre signalin değerlendirmek için kullanılabilir araçları kümesi genişletmektedirg gelişimi sırasında yollar ve özellikle gelişmekte olan kalp kapakçıkları için, immünohistokimyaya nicel bir iltifat sağlar. Bu teknik gelişim kardiyoloji değil, aynı zamanda sınırlı bir doku ihtiva eden bölümleri ile ilgili erken evre embriyoları ile çalışan tüm gelişim biyolojistlere sadece yararlı olacaktır.

<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.

Bu hazırlık tekniği kullanarak, E13.5 embriyolar tek aort kapak bölgelerinde fosforile Smad 1,5,8 (pSmad) tespit başardık. Şekil 1A&#39;da gösterildiği gibi, hatta bir tek valf bölgesinden izole edilmiş protein bir hafif pSmad grubu tespit etmek için yeterlidir. Sinyal yoğunluğu bir araya getirilmiş, kapak bölgelerinin sayısı artar. Önemli olarak, pSmad / β-aktin oran, farklı örnek boyutları (Şekil 1C) boyunca hemen hemen sabit kalır. Nedeniyle mevcut...

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.

Gelişmekte Embriyonik Fare Kalp Vana Bölgesi&#39;nde Protein İzolasyonu

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

Research

JoVE Journal

Biology

9.3K Görüntüleme.  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.

RNA veya Protein Ekstraksiyonu Özgül Beyin Bölgesi&#39;nden Rat Beyin Mikro-disseksiyon

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

Biyoloji

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.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

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.

Embriyonik Fare Böbrek Nefrojenik Zone Hücreleri İzolasyon ve Kültür

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.

Zebra balığı Embriyonik Kalp Dissected, immün

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.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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;

Fare Kalpten İlköğretim Melanocyte gibi Hücreleri Ayırma

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.

Yeni Bir<em&gt; Ex vivo</emEmbriyonik Fare Kalp için&gt; Kültür Yöntemi

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.

Embriyonik Ventriküler Endotel Hücre İzolasyonu

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.

Embriyonik Fare Kalpler Gelişmekte Hücre Etiketleme ve Enjeksiyon

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.

Murin Vana Endotel Hücreleri İzolasyon

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