Biz okuma ve yazma düzenleme için Brad Lackford teşekkür ederim. Bu araştırma, Çevre Sağlığı Bilimleri, Sağlık İntramural Araştırma Programı Z01ES102745 National Institutes (GH) Ulusal Enstitüsü tarafından desteklenmiştir.

1. Oct4GiP Fare ES Hücre Bakım Oct4GiP hücreleri nazikçe Dr Austin Smith tarafından sağlandı. Onlar bir Oct4-GFPiresPac transgen 4,5 taşıyan 129/Ola farelerden elde edilmiştir. Bunlar ESGRO tam artı klonal dereceli orta (Millipore), ya da M15 ortamda jelatin kaplı doku kültür plakaları içinde muhafaza edilir: DMEM (Invitrogen)% 15 FBS, 1000 U / ml ESGRO (Millipore), 1x olmayan ile desteklenmiş temel amino asitler (Invitrogen), 1x EmbryoMax Nucleasides (Millipore), ve 10 uM β-merkaptoetanol. <ol><li> 30 dakika için oda sıcaklığında% 0.1 jelatin (Sigma) (0.1 ml jelatin çözeltisi / cm 2) ile kaplamak plakaları. </li><li> Plaka Oct4GiP ~ 2 jelatin kaplı plakalar hücreler x 10 4 / cm 2. </li><li> % 0.05 tripsin ile her iki günde bir hücre bölün. </li></ol> 2. Oct4GiP Hücrelerinde siRNA Transfeksiyon Oct4GiP reporter assay en convenien olduğunuM15 orta veya levha 384-iyi (Corning veya BD) - tly jelatin kaplı düz dipli 96 yürütülmektedir. Genel prosedürü Şekil 1&#39;de belirtilmiştir. <ol><li> siRNA-lipid kompleksleri montaj: <ol><li> 96-kuyucuklu plaklar içinde transfeksiyon için, U-alt siRNA-lipid kompleksler 96-kuyucuklu plaklar araya. </li><li> Her bir yanı içerisinde, 10 ul OptiMem (Invitrogen) ve 0.3 ul lipofektamin 2000 (Invitrogen) karıştırmak ve 5 dk için oda sıcaklığında inkübe edilir. </li><li> Lipid-OptiMem karışımına 5 pmol siRNA ekleyin ve bir 15 dakika daha inkübe edilir. SiRNA: 6ul: yağ oranı 100 pmol olduğunu. </li><li> Bir çok kanallı pipet yardımıyla jelatin kaplı düz alt plaka U-alt plaka OptiMem yılında siRNA-lipid kompleksleri aktarın. </li><li> 384 kuyucuklu plaklar içinde transfeksiyon için, 0.1 ul lipofektamin 2000 ve 10 ul OptiMem içinde 2 pmol siRNA kullanılarak siRNA-lipid kompleksleri hazırlanır. </li></ol></li></ol> Not: nihai konsantrasyon siRNATransfeksiyonlar 50 nm&#39;dir. 3-4 biyolojik yürütmek her siRNA transfeksiyon için çoğaltır. Düz alt jelatin kaplı plaka içine U-alt plaka ve tablet olarak siRNA-lipid kompleksleri ana karışımları ayarlayın. <ol start="2"><li> Oct4GiP hücre kaplama ve transfeksiyon: <ol><li> 96-kuyucuklu plaklar içinde transfeksiyon için, Oct4GiP hücreleri toplamak ve 9 x 10 4 hücre / ml taze M15 ortam içinde yeniden süspanse bunların. </li><li> Ayrıca, bir çok-kanallı pipet kullanılarak her biri için hücre süspansiyonu 100 ul ekleyin. </li><li> 3-5 kez aşağı yukarı pipeting tarafından iyi öncesi bölünüp siRNA-lipidler kompleksleri ile hücre süspansiyonu karıştırın. Farklı siRNA Transfeksiyonlar için ipuçları değiştirin. </li><li> 384 kuyucuklu plaklar, Pastör pipetiyle Oct4GiP hücrelerinde Transfeksiyon için 6 ila 10 x 4 hücre / ml her oyuğa ve tablet 30 ul hücre süspansiyonu. </li></ol></li></ol> Not: optimum hücre yoğunluğu, genellikle kaplama 3 x 10 5 hücre / cm 2, ancak may dramatik bir hücre büyüme veya canlılığını etkileyen siRNA&#39;lar için daha fazla optimizasyon gerektirir. Düşük yoğunluklu kaplama transfeksiyon sırasında kötü hücre sağkalım yol açacaktır. Yüksek kaplama yoğunluğu yüksek hücre izdiham bağlı farklılaşması nedeniyle yüksek arka yol açacaktır. Eğer 2-4 x 10 5 hücre / cm 2 arasında gerekli test kaplama yoğunluğu. <ol start="3"><li> Orta değişikliği: <ol><li> Çok kanallı aspiratör (Corning) veya vakum değnek (VP-Scientific) kullanılarak eski orta çıkarın. Plakalar altındaki hücreleri çizmemeye dikkatli olun. </li><li> Her gün çok kanallı pipet kullanarak taze ES hücre orta (384-de plaka için 96-plaka ve 30 ul 100 ul) ile hücrelerin besleyin. </li></ol></li></ol> 3. FACS analizi <ol><li> Hücre ayrışma: <ol><li> Dört gün transfeksiyondan sonra, çok kanallı aspiratör (Corning) veya vakum değnek (VP-Bilimsel) kullanarak, orta kaldırın. </li><li> 96 oyuklu plakalar için, 100μ ile bir kez hücreleri durulamal PBS. </li><li> Iyi çok kanallı pipet kullanarak her 25 ul% 0.25 tripsin ekleyin. </li><li> Ara sıra ajitasyon ile 5minutes için oda sıcaklığında inkübe edilir, ve görsel hücrelerin tam dekolmanı sağlamak için kontrol. </li><li> Tripsin inaktive ve tekrarlanan pipetleme tek bir hücre süspansiyonu olarak hücreleri ayırmak için% 10 FCS ile 90 ul PBS ekleyin. </li><li> 384-iyi plakalar için,% 10 FBS ile 30 ul PBS ile 10 ul tripsin ve soğutmadan hücreleri kesmek. </li></ol></li><li> FACS analizi: <ol><li> HTS ünitesi veya diğer benzer donanımlı FACS analizörü (örneğin Accuri veya Intellicyt gibi) ile donatılmış BD LSRII FACS analizörde GFP floresans analiz edin. </li><li> HTS üzerinde yüksek verimlilik modu kullanın ve hücre süspansiyonu 10 ul analiz. 1.0 x 10 4 hücrelere sayısı eşiği ayarlayın. </li><li> Ileri vs yan scatter plot hücreleri yakalamak için PMT voltaj ve eşik ayarlayın. Forw canlı hücre popülasyonu için Kapısıard vs yan scatter plot. </li><li> GFP kanal için bir histogram grafiği oluşturun. Hücrelerinin ~% 10 sahte veya kontrol siRNA-transfekte edilmiş hücrelerde GFP-negatif olarak görünür şekilde GFP kanalda kapısı ayarlanır. </li><li> % Farklılaştırılmış hücreler =% GFP-negatif: Her tedaviden% GFP-negatif hücreleri belirleyin. 2 kuyruklu t-testi kullanılarak deney-kontrol-siRNA transfeksiyonlar arasında% Farklılaştırılmış hücreleri karşılaştırın ve p değerleri &lt;0.01 ile pozitif hit puan. </li></ol></li></ol> 4. Temsilcisi Sonuçlar Oct4, Nanog ve Sox2 ES hücre kendini yenileme 6,7,9-11 bakımında kritik bir rol oynayan üç gen vardır. Şekil Oct4GiP reporter assay kolaylıkla bu faktörlerin susturmak kaynaklanan farklılaşma tespit edebilen 2 gösteri Oct4GiP ES hücreleri. Şekil 2A ES olarak bakımı yapıldığında Oct4GiP ES hücrelerinin GFP pozitif olduğunu göstermektedir<html> . Şekil 2B hücreleri ileri vs yan dağılım çizim ve kontrolü-ya da-Oct4 siRNA&#39;lar ile transfekte edilmiş hücrelerin Oct4GiP GFP kanalının histogram gösterir. Ölü hücreleri ve enkaz GFP-negatif ve arka plan artacak gibi, ileri vs yan scatter plot canlı hücreler için kapısı gereklidir. Diğer taraftan, mümkün hücre kümeleri dışlamak için ayrımcılık eş her zaman gerekli değildir. Kontrol-siRNA transfekte hücrelerinde, hücrelerin büyük çoğunluğu GFP-pozitif olmalıdır. Bariz GFP-negatif popülasyonları kontrol siRNA transfekte kuyularda varsa, başlangıç ​​Oct4GiP hücreleri veya transfeksiyon prosedürü tehlikeye olabilir ve sonuç yorumlanabilir olmayabilir. Şekil 2C den% Farklılaştırılmış hücreleri (% Farklılaştırılmış hücreleri =% GFP-negatif hücreler) grafik gösterir kontrol, Oct4-, Nanog-ve Sox2-siRNA transfekte hücreler. <html> d/3987/3987fig1.jpg &quot;/&gt; Şekil 1.. Oct4GiP reporter assay Taslağı. <img alt="Şekil 2" src="/files/ftp_upload/3987/3987fig2.jpg" /> Şekil 2. Oct4GiP reporter assay Nanog, Oct4 veya Sox2 suskunluğu neden ES hücre farklılaşması algılayabilir. A) E14Tg2a (yabani tip, siyah çizgi) ve Oct4GiP (yeşil hat) hücreler FACS analiz edildi. GFP-kanalının histogram Oct4GiP hücreleri GFP-pozitif olduğu. B) Oct4GiP hücreleri Kontrol-ya da-Oct4 siRNA ile 96-kuyucuklu plaklar içinde transfekte edilmiş ve transfeksiyondan FACS 4 gün sonra incelendi gösterir. İleri Transfekte edilmiş hücrelerin GFP-kanalının yan saçılım histogram vs gösterilmiştir. C) Oct4GiP hücreleri ile transfekte edildi Kontrol-,-Nanog, Oct4-, veya-Sox2 siRNA. % Farklılaştırılmış Hücreler% 4 gün transfeksiyondan sonra hücreler GFP-negatif tespit edildi ve + / ortalama olarak çizilmiştir - ortalama standart hata (n = 4).f = &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot; target = &quot;_blank&quot;&gt; büyük rakamı görmek için buraya tıklayın.

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Çıkar çatışması ilan etti.

Yukarıda tarif Oct4GiP reporter assay kantitatif kendini yenileme vs farklılaşma derecesini ölçebilirsiniz. Böyle morfolojisi tabanlı 12 ve proliferasyonu / deneyleri yaşayabilirliği bazlı gibi diğer yöntemler kullanılabilir, ile karşılaştırıldığında, daha yüksek verim ve hassasiyet yanı sıra ES hücresi durumunu daha doğrudan bir ölçüm sunmaktadır. Bu yüzden de, büyük ölçekli ekranlar ve genetik epistasi analizi için son derece uygundur. Nitekim, biz ve diğerleri başarıy...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> ESGRO tam artı klonal notu orta </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (Yüksek glukoz 1X) </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> % 0.25 tripsin-EDTA </td><td> Invitrogen </td><td> 25200 </td></tr><tr><td> Lipofektamin 2000 </td><td> Invitrogen </td><td> 1001817 </td></tr><tr><td> OPTI-MEM (serum orta azaltmak) </td><td> Invitrogen </td><td> 31985 </td></tr><tr><td> ESGRO mLIF (10 units/1ml 7) </td><td> Millipore </td><td> DAM1776540 </td></tr><tr><td> MEM NEAA (Non-Essential Amino Asitler) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> ES hücre için 100x nükleosidlerin </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> 2-merkaptoetanol </td><td> Sigma </td><td> M7522-100ml </td></tr><tr><td> ES-nitelikli fetal sığır serumu </td><td> Invitrogen </td><td> 10437 </td></tr><tr><td> Nanog siRNA </td><td> Invitrogen </td><td> MSS231181 </td></tr><tr><td> Oct4 siRNA </td><td> Dharmacon </td><td> D-046256-02 </td></tr><tr><td> Sox2 siRNA </td><td> Dharmacon </td><td> M-058489-01 </td></tr></tbody></table> Kontrol siRNA: Dharamcon tarafından sentezlenen ateşböceği lusiferaz (5&#39;-CGTACGCGGAATACTTCGA) hedef siRNA dubleks.

oct4gip reporter assay to study genes that regulate mouse embryonic stem cell maintenance and self-renewal

Pluripotency ve kendini yenileme embriyonik kök hücreleri iki belirleyici özellikleri (ES hücreleri) vardır. Altta yatan moleküler mekanizmayı anlamak oldukça gelişimsel biyoloji çalışmaları, hastalık modelleme, ilaç keşfi ve rejeneratif tıbbın (1,2 gözden) ES hücrelerin kullanımı kolaylaştıracaktır. ES hücre bakım ve kendini yenileme romanı düzenleyicilerinin belirlenmesi ve tanımlanması hızlandırmak için, kantitatif Oct4GiP hücreleri kullanarak fare 3 ES hücrelerinin kendini yenileme durumunu ölçmek için floresans muhabiri bazlı analiz geliştirdi. Oct4GiP hücreleri 4,5 Oct4 geninin promotor bölgesinin kontrolü altında yeşil floresan proteininin (GFP) ifade eder. Oct4 farklılaşma 6,7 sırasında ES hücre kendini yenileme için gereklidir ve son derece ES hücreleri ile ifade edilir ve hızlı bir şekilde aşağı düzenlenir. Bunun sonucu olarak, raportör hücrelerinde GFP tanımı ve floresans aslına ilişkilendirenES hücresi kimlik 5 ve floresans-aktive hücre ayırma ile (FACS) analizi yakından tek hücre seviyesinde 3,8 azından hücrelerinin kendini yenileme durumunu kontrol etmek için kullanılabilir. RNAi ile birleştiğinde, Oct4GiP reporter assay hızlı ES hücre bakım ve kendini yenileme 3,8 regülatörleri belirlemek ve incelemek için kullanılabilir. Kendini yenileme içlin diğer yöntemlerle karşılaştırıldığında, daha fazla, uygun duyarlı, kantitatif, ve daha düşük maliyetli taşımaktadır. Bu tür yüksek verimli ekranlar veya genetik epistasi analiz gibi büyük ölçekli çalışmalar için veya plakaları 384-iyi - 96 yapılabilir. Son olarak, diğer soy özel muhabiri ES hücre hatları kullanılarak, biz burada açıklamak tahlil de ES hücre farklılaşması sırasında kaderi özellikleri incelemek için modifiye edilebilir.

Watch this Scientific Journal Video about Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal at JoVE.com

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Biz hızla belirlemek ve fare embriyonik kök hücre bakım ve kendini yenileme düzenleyen genlerin karakterize bir floresan reporter assay açıklar.

Fare Embriyonik Kök Hücre Bakım ve Kendini yenileme Regulate Genler Okumak Oct4GiP Reporter Assay

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

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10.4K Görüntüleme.  National Institute of Environmental Health Sciences. Biz hızla belirlemek ve fare embriyonik kök hücre bakım ve kendini yenileme düzenleyen genlerin karakterize bir floresan reporter assay açıklar.

Video: Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Biyoloji

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro ve in vivo Biyoparlaklık Reporter Gen Görüntüleme İnsan Embriyonik Kök Hücreleri

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

İnsan Embriyonik Kök Hücre Kültürü ve Bakım

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

Kendini yenileme LIF Yokluğunda Mesc Destek SC1 (Pluripotin) kullanımı

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

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 ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Embriyonik Ekstremite Cilt damarları Tüm montaj İmmünohistokimyasal Analizi: Embriyo vasküler dallanma Morphogenesis Eğitim Model Sistemi

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Kardiyojenik İndüksiyon Verimlilik miktarının için modifiye Fare Embriyonik Kök Hücre tabanlı Testi

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Erken evre Fare Embriyolar kullanarak Doku özel Genler Kromatin Immunoprecipitation Testi

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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 ...