Biz kritik yazının okuma ve finansman desteği için NIH (hibe # R01HL061656) için Andrea Portbury teşekkür etmek istiyorum.

1. Embriyonik Kalpler eksize <ol><li> Onaylanmış bir ötenazi tekniği kullanarak istenen embriyonik gün bir zamanlı hamile fare Euthanize. Tüm deneyler Chapel Hill&#39;deki North Carolina Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmıştır. </li><li> Liberal diseksiyon önce% 70 etanol ile kadın sprey. Uterin boynuz almak ve tüketim için dişinin karın boşluğuna açın. </li><li> Soğuk 1x fosfat tamponlu salin (PBS) ve gerektiğinde durulama içeren bir Petri kabındaki uterin horn yerleştirin. Ekli embriyonik kesesi ortaya çıkarmak için orta hat üzerinden uterin horn açın kesin. </li><li> Bir embriyonik kese açık kesin ve bir embriyo almak. Soğuk PBS ile ikinci bir Petri tabağına yerleştirin embriyo. Buz rahim boynuz ile Petri kabı dönün. </li><li> Göğüs duvarı erişimi artırmak için embriyo başını kesmek. Genotiplendirme gerekli ise, çıkarın ve buz üzerinde yerleştirilen bir Eppendorf tüp kuyruk kaydedin. </li><li> Ilesırtında embriyo, kalp ve akciğer görselleştirmek için göğüs duvarı açın kesti. Sahne bağlı olarak, göğüs duvarı şeffaf olabilir. </li><li> Dikkatlice forseps kullanarak kalp kaldırın ve aşağıdaki damarları kesti. Daha sonra, kalp boşaltmak için büyük damarların üzerinde kesti. Gerekirse, yabancı dokuları çıkarın. </li><li> Soğuk PBS ile doldurulmuş, 24 kuyulu bir çanak iyi birinin kalp koyun ve buz üzerine kabı yerleştirin. </li><li> Kalpleri tüm embriyolardan eksize edilmiştir kadar önceki adımları tekrarlayın. </li></ol> 2. Kültür Kurulum <ol><li> Laminar bir başlık olarak, 1:1 oranında soğuk Matrigel ve istenen kültür ortamı (Dulbecco&#39;nun Modifiye Edilmiş Eagle Ortamı (DMEM), örneğin,% 10 fetal sığır serumu (FBS)) birleştirir. Ön ısıtma kültür ortamı yok. </li><li> Buz, aynı zamanda buz üzerinde muhafaza edilir ve bir 24-çukurlu kültür çanağı bir kuyuya seyreltilmiş bir çözelti pipetle 500-1.000 ul seyreltilmiş Matrigel üzerinde tutulması. </li><li> Kaputu, dikkatli almak ve eksize yerMatrigel içeren kültür çanak bir de kalp buz üzerinde kültür çanak tutarken. </li><li> Gömülü kalp yönü önemli ise: Liberal% 70 etanol ile ters bir mikroskop ve çevresindeki alan sprey. Kaputu, ise hala buz üzerinde Matrigel içeren kültür plaka bir kuyuda eksize kalp yerleştirin. Daha sonra, bir mikroskop plaka yerleştirin ve hızlı bir şekilde yönlendirmek eksize kalp Matrigel katılaşmaya başlar. </li><li> Bir nemlendirilmiş 37 ° C inkübatör (% 5 CO2) içinde kültür tabağına yerleştirin ve Matrigel yaklaşık 30 dakika boyunca katılaşmasını sağlar. </li><li> Iyi bir kalp içeren her 1 ml önceden ısıtılmış kültür ortamı ekleyin. </li><li> Kalpler en az dört gün boyunca kültür kalabilir. Kültür ortamı her iki günde bir değiştirilmesi tavsiye edilir. </li></ol> 3. Tespit ve Görselleştirme <ol><li> Arzu edildiği takdirde, canlı kalp görselleştirmek için (örneğin SYTO-16), bir floresan boya canlı hücre ekleyin. Fotoğraf olacakbu gömülü edildiği konumuna dayalı görünür kalbin tarafına sınırlı. </li><li> Sonrası kültür analizleri (örneğin tüm montaj görüntüleme veya kesit) yapılacak ise, kültür ortamı çıkarın ve soğuk% 4 formaldehit ile değiştirin. 30 dakika boyunca buz üzerinde tabağına yerleştirin. </li><li> Soğuk formaldehit ve buz Matrigel dağılması teşvik sonra, taze formaldehit ile (ve Matrigel) fiksatif yerine ve 4 gece kalpleri ° C düzeltmek </li><li> 4 ° C kadar daha analizler de tampon (örneğin PBS veya Tris) ve mağaza ile kalpleri yıkayın. </li></ol>

<ol>
	<li>Tu, S., Chi, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+in+cardiac+development+and+congenital+heart+birth+defects.">Zebrafish models in cardiac development and congenital heart birth defects.</a> Differentiation. 84, 4-16 (2012).</li><li>Lavine, K. J., 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=Fibroblast+growth+factor+signals+regulate+a+wave+of+Hedgehog+activation+that+is+essential+for+coronary+vascular+development.">Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development.</a> Genes Dev. 20, 1651-1666 (2006).</li><li>Red-Horse, K., Ueno, H., Weissman, I. L., Krasnow, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+arteries+form+by+developmental+reprogramming+of+venous+cells.">Coronary arteries form by developmental reprogramming of venous cells.</a> Nature. 464, 549-553 (2010).</li><li>Stuckmann, I., Evans, S., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+and+retinoic+acid,+secreted+from+the+epicardium,+are+required+for+cardiac+myocyte+proliferation.">Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation.</a> Dev. Biol. 255, 334-349 (2003).</li><li>Zhang, J., 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=The+FGF-BMP+signaling+axis+regulates+outflow+tract+valve+primordium+formation+by+promoting+cushion+neural+crest+cell+differentiation.">The FGF-BMP signaling axis regulates outflow tract valve primordium formation by promoting cushion neural crest cell differentiation.</a> Circ. Res. 107, 1209-1219 (2010).</li><li>Pillekamp, F., 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=Establishment+and+characterization+of+a+mouse+embryonic+heart+slice+preparation.">Establishment and characterization of a mouse embryonic heart slice preparation.</a> Cellular Physiology and Biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology. 16, 127-132 (2005).</li><li>Habeler, W., Peschanski, M., Monville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+heart+slices+for+cell+transplantation+and+physiological+studies.">Organotypic heart slices for cell transplantation and physiological studies.</a> Organogenesis. 5, 62-66 (2009).</li><li>Janssen, P. M., Lehnart, S. E., Prestle, J., Hasenfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+contractile+characteristics+of+human+myocardium+in+multi-day+cell+culture.">Preservation of contractile characteristics of human myocardium in multi-day cell culture.</a> J. Mol. Cell Cardiol. 31, 1419-1427 (1999).</li><li>Brandenburger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+culture+from+human+adult+ventricular+myocardium.">Organotypic slice culture from human adult ventricular myocardium.</a> Cardiovasc. Res. 93, 50-59 (2012).</li><li>Weaver, M., Dunn, N. R., Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp4+and+Fgf10+play+opposing+roles+during+lung+bud+morphogenesis.">Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis.</a> Development. , 127-2695 (2000).</li><li>Viragh, S., Challice, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+epicardium+and+the+embryonic+myocardial+circulation+in+the+mouse.">The origin of the epicardium and the embryonic myocardial circulation in the mouse.</a> Anat. Rec. 201, 157-168 (1981).</li><li>Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiac Development. , (2007).</li><li>Arima, 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=Angiogenic+morphogenesis+driven+by+dynamic+and+heterogeneous+collective+endothelial+cell+movement.">Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement.</a> Development. 138, 4763-4776 (2011).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Mevcut kültür sistemi embriyonik fare kalp çalışmalar için önemli avantajlar teşkil etmektedir. Bu kültür sistemi bile kültüründe dört gün sonra, nekroz sınırlı işaretleri ile miyokardiyal kasılma ve koroner pleksus, korur. Bundan başka, yan-katı matris kültürü sırasında yerinde kaplanmış boncuklar tutmak için, aynı zamanda sözleşmesi esneklik sağlayan ve geliştirme sırasında kalbin üç boyutlu morfolojisi korumak için yeterli destek sağlar. Bu destek rağmen, bu matris, canlı ka...

Son yıllarda, transgenik fare gelişimi kalp kusurları eğitim için baskın model sistem olmuştur. Ancak, Zebra balığı gibi diğer model organizmalar, fare üzerinde önemli avantajlara sahip kanıtlanmıştır. Zebra balığı üç ana avantajları embriyoların erişim kolaylığı için, yumurta dış döşeme olan, kalp gelişim kolay görüntüleme sağlayan embriyoların optik şeffaflık, ve küçük moleküler tedaviler uygulama kolaylığı embriyo gelişimi modüle 1. Böylece, bir embriyonik organı eski rahimde büyüme izin veren bir kültür tekniği gelişimi, en azından kısmen, sınırlama anda transgenik farenin gelişim süreçleri çalışan araştırmacılar tarafından yaşanan, etkisiz hale getirebilir. Ex vivo kültür sistemleri kardiyak dif ne kadar küçük moleküller ve analiz ile muamele izin piliç ve fare embriyo hem de geliştirilmiştirKalbin lara bölgelerinde 2-6 iletişim. Bütün fare kalbinde bir kültür için, kalpler embriyonik için embriyolarından alınmış (E) yaş 12.5 sallanan 2,3,5 olan veya olmayan kültür ortamı içinde yerleştirilebilir. Bu tekniği kullanarak, embriyonik kalpleri başarıyla E13.5 en denklik için inkübe edilmiş ve sallanan ile kültür kalpleri sürece üç gün (E10.5 başlayarak) 3 olarak muhafaza edilmiş. Ancak, hiçbir çalışma eski embriyolardan kalplerin başarılı kültür bildirdin. Aynı şekilde, kurtarma deneyler, kültür ortamı 2 ile genel olarak terapötik ajanı uygulayarak sınırlı olmuştur. Kalpler, eksize gömülü ve bir vibratome kullanarak kesitli edildiği bir dilim kültür sistemi, aynı zamanda E12.5 fare kalpleri ve Hamburger-Hamilton aşamasında 36 (yaklaşık E16 fare) civciv kalpleri her iki genç kalpler, için kullanılmıştır Bu doğum sonrası ve yetişkin fare saat olarak 2,4,6, ve eski kalpler,earts ve yetişkin insan kalpleri 7,8. Embriyonik analizleri genellikle 150 mikron kalınlığında kesitler 2,4 kullanılan olsa da, kesit kalınlığı oksijen yoksunluğu 8 kanıtı olmadan büyük 500 olarak mikron olabilir. Bu dilim kültürleri en dilimleri bu dönemde 9 boyunca kasılma bakımı ile, kültür iki ay sürece devam edilmiştir. Izole kardiyomiyositlerde çalışmalar ile karşılaştırıldığında, bu dilim kültürler komşu hücre tipleri ile kardiyomiyositlerinin ko-kültür izin ve ex vivo analizi için yararlı bir yöntem sağlar. Bununla birlikte, bu kültürler sadece kültür ortamı içinde bir kalp (örneğin, bir vibratome üzerinde kesit için canlı kalp gömme) yerleştirerek daha ayrıntılı bir set-up gerektirir ve herhangi bir analiz açıkça bölüm içindeki kalbin kısmı ile sınırlıdır. Sınırlamaları kültür embriyonik fare kalpleri ve transgenik farelerin ava zenginliği için yukarıda açıklanan verilençalışma için ilable, biz Weaver tarafından geliştirilen bir ex vivo akciğer kültür sistemi benzer bir ex vivo fare kalp kültür sistemi geliştirdi, ve ark. 10. Bizim kültür sistemi bütün embriyonik fare kalplerinde yeniden koroner dolaşım uzun süreli kültür ve görselleştirme izin verir. Buna ek olarak, Matrigel kullanımı, böylece terapötik maddeler ile bir lokal tedavi sağlayan, boncuk kalp yakın bir yerde tutulabilir sağlar. Bu deneyler, örneğin koroner arter oluşumu gibi bir proses ile ilgili belirli bir tedavinin etkisini karşılaştırmak için farklı gelişimsel zaman noktalarında gerçekleştirilebilir. Küçük moleküllerin Matrigel difüzyonuna olduğundan, bu kültür sistemi, aynı zamanda belirli bir hücre-hücre temas bazı gelişim süreçleri için gerekli ya da başka bir bölgeden parakrin sinyal olup olmadığını olup olmadığını belirlemek için birbirlerine yakın kalp kültürü parçalara bölgeleri için kullanılabilir gerekli değildir. Bu kültür sisteminispeten basit ve kesit kültürü sisteminden farklı olarak, temel kültür çoğu laboratuar reaktifler ve hazır olan set-up kullanır. Kısaca, kalpler kesilen embriyonik bir yarı-katı bir destek içerir, seyreltik Matrigel kültürlenmiştir. Bu destek, kalbin büzülmesine izin verirken, kalbin üç boyutlu morfolojisi korumak için yeterlidir. Bu sistemi kullanarak, eski bir fare embriyoları (E14.5-E16.5) gelen bütün kalpleri dört gün için kültür korunabilir. Tüm koroner pleksus dilim kültürlerde farklı, tutulan, bu nedenle kalbin farklı bölgelerinden meydana gelen sinyal ipuçları mevcut kalır. Ayrıca, canlı hücre floresan boyalar canlı kalbin görüntülenmesi izin vermek için, Matrigel nüfuz edebilir, ve protein ile konjüge boncuk lokalize bir sinyal kaynağı sağlamak için kalp yakın yerleştirilebilir. Birlikte, bu avantajları bu teknik embriyo gelişim süreçlerini incelemek için ideal bir yöntem yapmakic fare kalp.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Timed-pregnant mice</td>
 <td></td>
 <td></td>
 <td>To be dissected at the embryonic stage of interest</td>
 </tr>
 <tr>
 <td>PBS (1x)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Cellgro </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Sigma-Aldrich </td>
 <td>F2442 </td>
 <td></td>
 </tr>
 <tr>
 <td>Growth factor-reduced Matrigel</td>
 <td>BD Bioscience </td>
 <td>356231 </td>
 <td></td>
 </tr>
 <tr>
 <td>Syto-16</td>
 <td>Invitrogen </td>
 <td>S7578 </td>
 <td>Used as directed in 13 </td>
 </tr>
 <tr>
 <td>24-well culture plate</td>
 <td>Fisher Scientific</td>
 <td>07-200-84</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Stereoscopic microscope</td>
 <td>Nikon </td>
 <td>SMZ645</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture incubator</td>
 <td>Thermo </td>
 <td>3110 </td>
 <td></td>
 </tr>
</tbody>
 </table>

a novel ex vivo culture method for the embryonic mouse heart

Fare gelişim çalışmaları gebelik sırasında embriyonun erişilememesi tarafından engellenmektedir. Bu nedenle, protokoller izole etmek ve ilgi kültürü, tek tek organların hem de geliştirme değişiklikler ve görselleştirmek sağlayan yeni tedavi stratejileri için bir yöntem sağlamak üzere gereklidir. Gebeliğin son aşamalarında embriyonik kalbin uzun vadeli bir kültür teşvik etmek için, eksize kalp, yarı-katı, seyreltik Matrigel kültüre edildiği bir protokolü geliştirilmiştir. Bu yüzey üç boyutlu yapısını korumak için yeterli destek sağlar, ancak devam eden daralma izin verecek kadar esnektir. Kısaca, kalpler embriyo çıkarıldı ve soğuk Matrigel karışımı yerleştirilir büyüme ortamı ile 1:01 seyreltilmiştir. Seyreltilmiş Matrigel katılaşmasından sonra, büyüme ortamı kültür tabağına ilave edilir. Embriyonik gün 16,5 gibi geç çıkarıldı Kalpler dört gün sonrası diseksiyon için geçerli idi. Koroner pleksus Analiz Bu yöntem, etmediğini göstermektedirkoroner damar gelişimi bozabilir. Böylece, embriyonik kalplerin uzun dönemli kültür için yeni bir yöntem sunmaktadır.

Bu tekniği kullanarak, kalp üç boyutlu morfoloji korur ve canlı kalır, olarak devam kasılmalar (Film 1) ile gösterilir. Bu kasılmalar ventrikül daha kulakçıklar sürekli olarak daha belirgindir. Kültür ardından, kupa sabit ve işlenmiş özel işaretleyici ifade veya yapıları incelemek için immünhistokimya veya histoloji biri için. Şekil 1A sabit, 24 saat süre ile kültüre olan bir embriyonik fare kalp ventriküller ve büyük arterlerin taban gösterir ve için i?...

Watch this Scientific Journal Video about A Novel Ex vivo Culture Method for the Embryonic Mouse Heart at JoVE.com

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Fare gelişim çalışmaları gebelik sırasında embriyonun erişilememesi tarafından engellenmektedir. Gebeliğin son aşamalarında embriyonik kalbin uzun vadeli bir kültür teşvik etmek için, eksize kalp, yarı-katı, seyreltik Matrigel kültüre edildiği bir protokolü geliştirilmiştir.

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 ...

a-novel-ex-vivo-culture-method-for-the-embryonic-mouse-heart

Research

JoVE Journal

Biology

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

13.2K Görüntüleme.  University of North Carolina at Chapel Hill. Fare gelişim çalışmaları gebelik sırasında embriyonun erişilememesi tarafından engellenmektedir. Gebeliğin son aşamalarında embriyonik kalbin uzun vadeli bir kültür teşvik etmek için, eksize kalp, yarı-katı, seyreltik Matrigel kültüre edildiği bir protokolü geliştirilmiştir.

Video: A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biyoloji

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

Fare Retina Ganglion Hücreleri Gen İfadesi utero ve ex vivo Elektroporasyon

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Fare Embriyonik Akciğer Kültür, Dallanma Moleküler Mekanizmalar değerlendirin Bir Sistem

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

İnsan bağırsak mukozasının eksplantlar Kültür ve Polarize Uyarım için Yeni Bir Yöntem

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

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 ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Serumsuz Tüm Embriyo Kültür Sistemde Fare Embriyonik Development

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Fare Embriyonik Cilt ve Melanoblast Göç Canlı görüntüleme ex vivo Kültür

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

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.

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 ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

c-FOS Protein Immünohistolojik Algılama: Belirli Fizyolojik Yanıtları ilgilendim Merkez Pathways bir Marker Olarak Faydalı Aracı<em&gt; İn Vivo</em&gt; ve<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...