Nous tenons à remercier Brad Lackford pour la lecture et la relecture du manuscrit. Cette recherche a été financée par l&#39;Institut national de l&#39;environnement Sciences de la santé, les National Institutes of Health Research Programme intra-muros Z01ES102745 (à GH).

1. Oct4GiP souris maintien des cellules ES Cellules Oct4GiP ont été aimablement fournies par le Dr Austin Smith. Elles ont été calculées à partir des souris portant un transgène 129/Ola Oct4-GFPiresPac 4,5. Ils sont maintenus dans de la gélatine recouvertes de plaques de culture tissulaire dans le ESGRO qualité moyenne, plus complet clonale (Millipore), ou dans le milieu M15: DMEM (Invitrogen) supplémenté avec 15% de FBS, 1000 U / ml ESGRO (Millipore), 1x non- acides aminés essentiels (Invitrogen), 1x Nucleasides EmbryoMax (Millipore), et 10 uM β-mercaptoéthanol. <ol><li> Manteau plaques avec 0,1% (Sigma) de la gélatine à la température ambiante pendant 30 minutes (0,1 ml de solution de gélatine / cm 2). </li><li> Oct4GiP cellules plaque en revêtues de gélatine à plaques ~ 2 x 10 4 / cm 2. </li><li> Fractionner les cellules tous les deux jours avec de la trypsine 0,05%. </li></ol> 2. transfection de siRNA dans les cellules Oct4GiP Le dosage est le plus journaliste Oct4GiP convenienTLY réalisée dans de la gélatine enduit fond plat 96 - ou 384 puits plaques (Corning ou BD) dans le milieu M15. La procédure générale est décrite dans la figure 1. <ol><li> siRNA-lipidique de montage complexes: <ol><li> Pour la transfection dans des plaques 96 puits, assembler siARN-lipidiques complexes dans U-bas plaques à 96 puits. </li><li> Dans chaque puits, mélanger 10 pi OptiMEM (Invitrogen) et 0,3 ul Lipofectamine 2000 (Invitrogen) et incuber à température ambiante pendant 5 min. </li><li> Ajouter 5 siARN pmol au mélange lipidique OptiMEM et incuber pendant 15 minutes. L&#39;ARNsi: lipide est de 100 pmol: 6UL. </li><li> Transférer les complexes lipidiques dans siARN-OptiMEM de la plaque en U en bas de la gélatine à revêtement à fond plat plaque avec une pipette à canaux multiples. </li><li> Pour la transfection dans plaques à 384 puits, préparer les complexes siARN-lipidiques en utilisant 0,1 pl Lipofectamine 2000 et 2 pmol siARN dans 10 OptiMEM ul. </li></ol></li></ol> Remarque: La concentration finale en siARNles transfections est de 50 nM. Effectuer biologique 3-4 répétitions pour chaque transfection de siRNA. Mettre en place des mélanges maîtres des complexes siARN-lipidiques dans la plaque de fond en U et dans la partie aliquote à fond plat de gélatine plaque revêtue. <ol start="2"><li> Placage cellule Oct4GiP et la transfection: <ol><li> Pour la transfection dans des plaques 96 puits, collecter les cellules Oct4GiP et les remettre en suspension dans un milieu frais M15 à 9 x 10 4 cellules / ml. </li><li> Ajouter 100 ml de la suspension cellulaire dans chaque puits en utilisant une pipette multi-canaux. </li><li> Mélanger la suspension de cellules avec les pré-aliquotés ARNsi des lipides complexes dans le puits par pipetage de haut en bas 3-5 fois. Changer conseils pour transfections de siRNA différents. </li><li> Pour la transfection dans des plaques de 384 puits, les cellules Oct4GiP Resuspendre à 6 x 10 4 cellules / ml et aliquote de 30 pl de suspension cellulaire dans chaque puits. </li></ol></li></ol> Note: La densité optimale des cellules de placage est généralement de 3 x 10 5 cellules / cm 2, mais il may besoin une optimisation plus poussée des siRNA qui a considérablement influer sur la croissance ou la viabilité cellulaire. Faible densité de placage mènera à la survie des cellules pauvres lors de la transfection. Haute densité de placage mènera à fond élevé en raison de la différenciation cellulaire induite par la confluence de haut. Si nécessaire, densité de placage d&#39;essai entre 2-4 x 10 5 cellules / cm 2. <ol start="3"><li> Changement moyen: <ol><li> Retirer ancien milieu à l&#39;aide multi-canal d&#39;aspiration (Corning) ou sous vide baguette (VP-scientifique). Veillez à ne pas rayer les cellules au fond des assiettes. </li><li> Nourrir les cellules avec le milieu de cellules ES frais (100 pi pour plaque de 96 puits et 30 pi pour plaque 384 puits) en utilisant une pipette multi-canaux tous les jours. </li></ol></li></ol> 3. Analyse par FACS <ol><li> Dissociation cellulaire: <ol><li> Quatre jours après la transfection, éliminer le milieu de l&#39;aide multi-canal d&#39;aspiration (Corning) ou sous vide baguette (VP-scientifique). </li><li> Pour plaques à 96 puits, rincer les cellules une fois avec 100μl PBS. </li><li> Ajouter 25 trypsine pi 0,25% à chaque puits en utilisant une pipette à canaux multiples. </li><li> Incuber à température ambiante pendant 5 minutes avec agitation occasionnelle, et une inspection visuelle pour s&#39;assurer un complet détachement des cellules. </li><li> Ajouter 90 ul de PBS avec 10% de FBS pour inactiver la trypsine et à dissocier les cellules en une seule suspension cellulaire par pipetage répété. </li><li> Pour plaques 384 puits, dissocier les cellules avec 10 ul de trypsine et de trempe avec 30 ul de PBS avec 10% de FBS. </li></ol></li><li> Analyse FACS: <ol><li> Analyser la fluorescence de la GFP sur l&#39;analyseur BD FACS LSRII équipée avec l&#39;unité de HTS ou autre analyseur équipé de façon similaire FACS (comme Accuri ou Intellicyt). </li><li> Utilisez le mode haut débit sur le HTS et d&#39;analyser les 10 ul de la suspension cellulaire. Régler le seuil de comptage à 1,0 x 10 4 cellules. </li><li> Régler la tension de PMT et le seuil de capturer les cellules dans l&#39;intrigue vs côté-dispersion. Porte de la population de cellules vivantes dans le forward vs côte diagramme de dispersion. </li><li> Créer un histogramme pour le canal GFP. Réglez la porte dans le canal de la GFP de sorte que ~ 10% des cellules semblent être la GFP-négative dans les cellules fictives ou de contrôle-siARN transfectées. </li><li> Déterminer% GFP-négatives des cellules de chaque traitement: les cellules différenciées%% = GFP-cellules négatives. Comparer les cellules différenciées entre expérimentales% et de commande-siRNA transfections en utilisant 2-tailed test-t, et marquer les réponses positives avec des valeurs p &lt;0,01. </li></ol></li></ol> 4. Les résultats représentatifs Oct4, Nanog, et Sox2 ya trois gènes qui jouent un rôle crucial dans le maintien de cellules souches embryonnaires d&#39;auto-renouvellement 6,7,9-11. Figure 2 montre que le dosage journaliste Oct4GiP pouvons facilement détecter la différenciation provoquée par faire taire ces facteurs dans le Oct4GiP cellules ES. La figure 2A montre les cellules ES sont Oct4GiP GFP-positives lorsqu&#39;il est maintenu en tant que ES<html> cellules. Figure 2B montre le diagramme de dispersion de l&#39;avant par rapport à côté et l&#39;histogramme du canal GFP des cellules transfectées avec Oct4GiP le contrôle ou Oct4 des siRNA. Il est nécessaire à la porte des cellules vivantes dans le nuage de points avant vs côté, comme les cellules mortes et les débris sont GFP-négatif et va augmenter de fond. D&#39;autre part, la discrimination doublet d&#39;exclure amas de cellules possibles n&#39;est pas toujours nécessaire. Dans les cellules transfectées de contrôle-siRNA, la grande majorité des cellules doit être GFP-positives. Si évidentes GFP-négatifs populations sont présentes dans les puits de contrôle-siRNA transfectées, les cellules Oct4GiP de départ ou la procédure de transfection peut avoir été compromise et le résultat peut ne pas être interprétable. La figure 2C montre l&#39;histogramme de cellules différenciées (cellules%% =% différenciés GFP-négatifs cellules) de contrôle, Oct4-, Nanog-et-Sox2 siRNA cellules transfectées. <html> d/3987/3987fig1.jpg &quot;/&gt; Figure 1. Aperçu du test journaliste Oct4GiP. <img alt="Figure 2" src="/files/ftp_upload/3987/3987fig2.jpg" /> Figure 2. Dosage journaliste Oct4GiP peut détecter la différenciation des cellules ES causée par Nanog, Oct4, ou Sox2 silence. A) E14Tg2a (de type sauvage, ligne noire) et Oct4GiP (ligne verte), les cellules ont été analysées par FACS. Histogramme de la GFP-canal montre que les cellules sont GFP-Oct4GiP positif. B) les cellules ont été transfectées dans Oct4GiP plaques à 96 puits avec le contrôle ou Oct4-siARN et FACS analysé 4 jours après la transfection. Forward vs diagrammes de dispersion secondaires et les histogrammes de la GFP-canal des cellules transfectées sont indiquées. C) des cellules ont été transfectées avec Oct4GiP le contrôle-, Nanog-, Oct4-ou-Sox2 siARN. Les cellules différenciées% a été déterminée à partir du% GFP-négative cellules 4 jours après la transfection, et a été tracée sous forme de moyenne + / - erreur-type de la moyenne (n = 4).f = &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot; target = &quot;_blank&quot;&gt; Cliquez ici pour agrandir la figure.

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Pas de conflits d&#39;intérêt déclarés.

Le dosage journaliste Oct4GiP nous décrivons ci-dessus peut mesurer quantitativement l&#39;étendue de l&#39;auto-renouvellement vs différenciation. Comparé à d&#39;autres méthodes disponibles, tels que les 12 morphologie basée et la prolifération / viabilité à base de tests, il offre une sensibilité plus élevée et le débit, ainsi que d&#39;une mesure plus directe de l&#39;état de la cellule ES. Il est donc bien adapté pour les grands écrans et des analyses épistasie génétique. En effet, no...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Moyennes ESGRO année complète, ainsi que des clones </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (glucose à haute 1X) </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> 0,25% de trypsine-EDTA </td><td> Invitrogen </td><td> 25200 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 1001817 </td></tr><tr><td> OPTI-MEM (milieu sans sérum à réduire) </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-acides aminés essentiels) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Nucléosides 100x pour les cellules souches embryonnaires </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> Le 2-mercaptoéthanol </td><td> Sigma </td><td> M7522-100ml </td></tr><tr><td> ES-qualifiée sérum de veau fœtal </td><td> Invitrogen </td><td> 10437 </td></tr><tr><td> SiARN Nanog </td><td> Invitrogen </td><td> MSS231181 </td></tr><tr><td> Oct4 siARN </td><td> Dharmacon </td><td> D-046256-02 </td></tr><tr><td> SiARN Sox2 </td><td> Dharmacon </td><td> M-058489-01 </td></tr></tbody></table> SiARN contrôle: duplex siRNA ciblant la luciférase de luciole (5&#39;-CGTACGCGGAATACTTCGA) synthétisée par Dharamcon.

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

Pluripotence et l&#39;auto-renouvellement sont deux caractéristiques qui définissent les cellules souches embryonnaires (cellules ES). Comprendre le mécanisme moléculaire sous-jacent va grandement faciliter l&#39;utilisation de cellules souches embryonnaires pour des études de biologie du développement, la modélisation des maladies, la découverte de médicaments et la médecine régénératrice (examiné en 1,2). Pour accélérer l&#39;identification et la caractérisation de nouveaux régulateurs de l&#39;entretien des cellules ES et d&#39;auto-renouvellement, nous avons développé une fluorescence journaliste basé test pour mesurer quantitativement l&#39;état d&#39;auto-renouvellement des cellules ES de souris en utilisant les cellules Oct4GiP 3. Les cellules Oct4GiP exprimer la protéine fluorescente verte (GFP) sous le contrôle de la région promoteur du gène Oct4 4,5. Oct4 est nécessaire pour les cellules souches embryonnaires d&#39;auto-renouvellement, et est fortement exprimée dans les cellules ES et rapidement régulé à la baisse lors de la différenciation 6,7. En conséquence, expression de la GFP et de la fluorescence dans les cellules reporter fidèlement corréléeavec l&#39;identité des cellules ES 5, et le tri cellulaire activé par fluorescence (FACS) peut être utilisé pour suivre de près l&#39;état d&#39;auto-renouvellement des cellules au niveau unicellulaire 3,8. Couplé avec l&#39;ARNi, le dosage journaliste Oct4GiP peut être utilisé pour rapidement identifier et d&#39;étudier les régulateurs de l&#39;entretien des cellules ES et l&#39;auto-renouvellement 3,8. Comparé à d&#39;autres méthodes de dosage de l&#39;auto-renouvellement, il est plus commode, sensible, quantitative, et de moindre coût. Il peut être réalisé en 96 - ou 384 puits des plaques pour études à grande échelle tels que les écrans à haut débit ou d&#39;analyse épistasie génétique. Enfin, en utilisant d&#39;autres lignes spécifiques de lignée de cellules ES Reporter, le dosage que nous décrivons ici peuvent également être modifiées pour étudier les spécifications sort lors de la différenciation des cellules ES.

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

Nous décrivons un test de fluorescence journaliste d&#39;identifier rapidement et de caractériser les gènes qui régulent la souris de maintenance de cellules souches embryonnaires et l&#39;auto-renouvellement.

Essai Reporter Oct4GiP d&#39;étudier les gènes qui régulent la souris maintenance des cellules souches embryonnaires et l&#39;auto-renouvellement

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

oct4gip-reporter-assay-to-study-genes-that-regulate-mouse-embryonic

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Biology

10.4K vues.  National Institute of Environmental Health Sciences. Nous décrivons un test de fluorescence journaliste d'identifier rapidement et de caractériser les gènes qui régulent la souris de maintenance de cellules souches embryonnaires et l'auto-renouvellement.

Vidéo: 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.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

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 ...

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Biologie

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.

Dérivation des cellules souches embryonnaires humaines par 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 ...

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 et in vivo en bioluminescence gène rapporteur imagerie des cellules souches embryonnaires humaines

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.

Culture et entretien des cellules souches embryonnaires humaines

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.

L&#39;utilisation de SC1 (Pluripotin) pour soutenir MESC autorenouvellement en l&#39;absence de LIF

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.

L&#39;isolement et la dérivation de cellules germinales embryonnaires de souris

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

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.

Whole-Mont analyse immunohistochimique à la vascularisation cutanée Limb embryonnaires: un modèle pour l&#39;étude vasculaire morphogenèse de branchement de l&#39;embryon

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.

Mise à jour embryonnaires de souris des cellules souches basée Assay pour quantifier l&#39;efficacité d&#39;induction cardiogénique

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.

Assay immunoprécipitation de la chromatine des gènes spécifiques de tissus en utilisant des embryons de souris en début de

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.

Génération de souris dérivées de cellules souches pluripotentes induites

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