Wir bedanken uns bei Brad Lackford zum Lesen und Editieren des Manuskripts. Diese Arbeit wurde vom National Institute of Environmental Health Sciences, National Institutes of Health Research Program Intramural Z01ES102745 (GH) unterstützt.

1. Oct4GiP Maus-ES-Zell-Wartung Oct4GiP Zellen wurden freundlicherweise von Dr. Austin Smith zur Verfügung gestellt. Sie wurden von den Mäusen, die eine 129/Ola Oct4-Transgen GFPiresPac 4,5 abgeleitet. Sie werden in mit Gelatine beschichteten Gewebekulturplatten in der Esgro komplette plus klonale Grad Medium (Millipore), oder in der M15-Medium: DMEM (Invitrogen) mit 15% FBS, 1000 U / ml Esgro (Millipore), 1x Nicht-ergänzt essentiellen Aminosäuren (Invitrogen), 1x EmbryoMax Nucleasides (Millipore) und 10 &amp;mgr; M β-Mercaptoethanol. <ol><li> Mantel-Platten mit 0,1% Gelatine (Sigma) bei Raumtemperatur für 30 Minuten (0,1 ml Gelatinelösung / cm 2). </li><li> Tafel Oct4GiP Zellen in mit Gelatine beschichteten Platten bei ~ 2 x 10 4 / cm 2. </li><li> Teilen Sie die Zellen alle zwei Tage mit 0,05% Trypsin. </li></ol> 2. siRNA Transfektion in Oct4GiP Cells Die Oct4GiP Reporter-Assay ist am convenienoder 384-Well-Platten (Corning oder BD) in der M15 Medium - TLY in Gelatine beschichtete Flachboden-96 durchgeführt. Der gesamte Vorgang ist in Abbildung 1 dargestellt. <ol><li> siRNA-Lipid-Komplexen Montage: <ol><li> Für die Transfektion in 96-well Platten, montieren siRNA-Lipid-Komplexen in U-Boden 96-well Platten. </li><li> In jede Vertiefung, mischen 10 pl OptiMEM (Invitrogen) und 0,3 ul Lipofectamin 2000 (Invitrogen) und Inkubation bei Raumtemperatur für 5 min. </li><li> In 5 pmol siRNA an die Lipid-OptiMEM Mischung und Inkubation für weitere 15 Minuten. Die siRNA: Lipid-Verhältnis ist 100 pmol: 6UL. </li><li> Übertragen Sie die siRNA-Lipid-Komplexen in OptiMEM aus der U-Bodenplatte zur Gelatine beschichtete Flachboden-Platte mit einer Mehrkanal-Pipette. </li><li> Für die Transfektion in 384-Well Platten, bereiten Sie die siRNA-Lipid-Komplexen mit 0,1 pl Lipofectamin 2000 und 2 pmol siRNA in 10 ul OptiMEM. </li></ol></li></ol> Hinweis: Die endgültige siRNA-Konzentration inDie Transfektionen beträgt 50 Nm. Führen Sie 3-4 biologischen Wiederholungen für jede siRNA-Transfektion. Richten Sie Master Mischungen der siRNA-Lipid-Komplexen in der U-Bodenplatte und Aliquot in die Flachboden-Gelatine beschichtete Platte. <ol start="2"><li> Oct4GiP Zelle Plating und Transfektion: <ol><li> Für die Transfektion in 96-well Platten, sammeln die Oct4GiP Zellen resuspendieren und sie in frischem Medium M15 bis 9 x 10 4 Zellen / ml. </li><li> Geben Sie 100 ul der Zellsuspension in jede Vertiefung mit einer Mehrkanal-Pipette. </li><li> Mischen der Zellsuspension mit dem Standard-aliquotierte siRNA-Komplexe Lipide in der Vertiefung durch Pipettieren nach oben und unten 3-5 mal. Ändern Sie Tipps für die verschiedenen siRNA Transfektionen. </li><li> Für die Transfektion in 384-Well-Platten, resuspendieren Oct4GiP Zellen zu 6 x 10 4 Zellen / ml und 30 ul Aliquot Zellsuspension in jede Vertiefung. </li></ol></li></ol> Hinweis: Die optimale Zell-Beschichtungs-Dichte ist in der Regel 3 x 10 5 Zellen / cm 2, aber es may erfordern eine weitere Optimierung für siRNAs, die einen dramatischen Einfluss auf das Zellwachstum oder die Lebensfähigkeit. Niedrige Plattierungsdichte kann zu einer schlechten Überleben der Zellen während der Transfektion führen. Hohe Beschichtungs-Dichte führt zu hohen Hintergrund durch hohe Zellkonfluenz induzierte Differenzierung führen. Wenn nötig, Test Beschichtungs-Dichte zwischen 2-4 x 10 5 Zellen / cm 2. <ol start="3"><li> Mittlere Veränderung: <ol><li> Entfernen Sie alte Medium unter Verwendung von Multi-Channel-Aspirator (Corning) oder Vakuum-Zauberstab (VP-Scientific). Achten Sie darauf, um die Zellen an der Unterseite der Platten zu kratzen. </li><li> Führen Sie die Zellen mit frischem ES-Zell-Medium (100 ul für 96-Well-Platte und 30 ul für 384-Well-Platte) mit Mehrkanal-Pipette jeden Tag. </li></ol></li></ol> 3. FACS-Analyse <ol><li> Zelldissoziation: <ol><li> Vier Tage nach der Transfektion zu entfernen Medium mit Mehrkanal-Saugvorrichtung (Corning) oder Vakuum-Stab (VP-Scientific). </li><li> Für 96-well Platten, spülen Sie die Zellen einmal mit 100μl PBS. </li><li> In 25 ul 0,25% Trypsin in jede Vertiefung mit einer Mehrkanal-Pipette. </li><li> Inkubieren bei Raumtemperatur für 5 Minuten unter gelegentlichem Schütteln, und die Inaugenscheinnahme zur vollständigen Ablösung der Zellen zu gewährleisten. </li><li> In 90 ul PBS mit 10% FBS, um das Trypsin zu inaktivieren und distanzieren Zellen zu einer einzigen Zellsuspension durch wiederholtes Pipettieren. </li><li> Für 384-well Platten, distanzieren Zellen mit 10 ul Trypsin und Quench mit 30 ul PBS mit 10% FBS. </li></ol></li><li> FACS-Analyse: <ol><li> Analysieren Sie die GFP-Fluoreszenz auf der BD LSRII FACS-Analyzer mit dem HTS-Gerät oder einen anderen ähnlich ausgestatteten FACS-Analyser (wie Accuri oder Intellicyt) ausgestattet. </li><li> Verwenden Sie das High-Throughput-Modus auf der HTS und analysieren 10 ul Zellsuspension. Sie können die Anzahl Schwellenwert bis 1,0 x 10 4 Zellen. </li><li> Passen PMT-Spannung und Schwelle, um die Zellen in der Vorwärts-vs-Seite Streudiagramm zu erfassen. Tor für die Live-Zell-Population in der vorwARD vs side-Streudiagramm. </li><li> Erstellen Sie ein Histogramm Plot für die GFP-Kanal. Sie das Tor in der GFP-Kanal, so dass ~ 10% der Zellen zu sein GFP-negativen in den mock-Steuerelement oder-siRNA transfizierten Zellen zu sehen. </li><li> Bestimmen% GFP-negativen Zellen aus jeder Behandlung: Differenzierte Zellen% =% GFP-negativen Zellen. Vergleichen Sie die% Differenzierte Zellen zwischen Experimental-und Kontroll-siRNA-Transfektionen mit 2-tailed t-Test, und erreichen Sie die positive Treffer mit p-Werte &lt;0,01. </li></ol></li></ol> 4. Repräsentative Ergebnisse Oct4, Nanog und Sox2 sind drei Gene, die eine entscheidende Rolle spielen bei der Aufrechterhaltung der ES-Zell-Selbsterneuerung 6,7,9-11. Abbildung 2 zeigt, dass die Oct4GiP Reporter-Assay leicht erkennen kann, die Differenzierung durch Schweigen zu dieser Faktoren in der verursachte Oct4GiP ES-Zellen. 2A zeigt die Oct4GiP ES-Zellen sind GFP-positive, wenn sie als ES beibehalten<html> Zellen. 2B zeigt die Seite nach vorne vs Streudiagramm und das Histogramm des GFP-Kanal der Oct4GiP Zellen mit dem Kontroll-oder Oct4-siRNAs transfiziert. Es ist notwendig, Gate für lebende Zellen in der vorderen Seite vs Streudiagramm, wie die toten Zellen und Zelltrümmer zu GFP-negativ sind und Hintergrund zu erhöhen. Auf der anderen Seite, Wams, um mögliche Diskriminierungen Zellklumpen auszuschließen ist nicht immer erforderlich. In den Kontroll-siRNA transfizierten Zellen sollte die überwiegende Mehrheit der Zellen zu sein GFP-positiven. Wenn offensichtlich GFP-negativen Populationen in den Kontroll-siRNA transfiziert Brunnen sind, können die Start Oct4GiP Zellen oder die Transfektion Verfahren kompromittiert worden sind und das Ergebnis kann nicht interpretiert werden. 2C zeigt die Balkenanzeige von% Differenzierte Zellen (% Differenzierte Zellen =% GFP-negativen Zellen) von Kontroll-, Oct4-, Nanog-und Sox2-siRNA transfizierten Zellen. <html> d/3987/3987fig1.jpg &quot;/&gt; Abbildung 1. Gliederung des Oct4GiP Reporter-Assay. <img alt="2" src="/files/ftp_upload/3987/3987fig2.jpg" /> Abbildung 2. Oct4GiP Reporter-Assay kann erkennen, ES-Zell-Differenzierung durch Nanog, Oct4, Sox2 oder Silencing verursacht. A) E14Tg2a (Wild-Typ, schwarze Linie) und Oct4GiP (grüne Linie) Zellen wurden durch FACS analysiert. Histogramm der GFP-Kanal zeigt, dass Oct4GiP Zellen GFP-positiv sind. B) Oct4GiP Zellen wurden in 96-well Platten mit dem Control-oder Oct4-siRNA transfiziert und FACS analysiert 4 Tage nach der Transfektion. Vorwärts vs Seite Streudiagramme und Histogramme des GFP-Kanal der transfizierten Zellen gezeigt werden. C) Oct4GiP Zellen wurden mit der Steuer-, Nanog-, Oct4-oder siRNA-Sox2. Die% differenzierten Zellen vom% GFP-negativen Zellen 4 Tage nach der Transfektion bestimmt und wurde aufgetragen als Mittelwert + / - Standardfehler des Mittelwertes (n = 4).f = &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot; target = &quot;_blank&quot;&gt; Klicken Sie hier für eine größere Abbildung zu sehen.

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Die Oct4GiP Reporter-Assay beschreiben wir oben kann quantitativ messen das Ausmaß der Selbst-Erneuerung vs Differenzierung. Im Vergleich zu anderen verfügbaren Verfahren, wie die Morphologie-basierten 12 und Proliferation / Lebensfähigkeit-basierten Assays, bietet eine höhere Empfindlichkeit und Durchsatz sowie eine direkte Messung der ES-Zelle Zustand. Es wird daher auch für große Bildschirme und genetische Analyse Epistasie geeignet. In der Tat haben wir und andere erfolgreich das Oct4GiP Reporter-As...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Esgro komplette plus klonale Klasse Medium </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (high Glucose-1X) </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> 0,25% Trypsin-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 (Serum-Medium zu reduzieren) </td><td> Invitrogen </td><td> 31985 </td></tr><tr><td> Esgro mLIF (10 7 units/1ml) </td><td> Millipore </td><td> DAM1776540 </td></tr><tr><td> MEM NEAA (Nicht-essentielle Aminosäuren) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> 100x Nucleosides für ES-Zell- </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> 2-Mercaptoethanol </td><td> Sigma </td><td> M7522-100ml </td></tr><tr><td> ES-qualifizierte Rinderfötenserum </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-046.256-02 </td></tr><tr><td> Sox2 siRNA </td><td> Dharmacon </td><td> M-058.489-01 </td></tr></tbody></table> Kontroll-siRNA: siRNA-Duplex Targeting Glühwürmchen-Luciferase (5&#39;-CGTACGCGGAATACTTCGA) durch Dharamcon synthetisiert.

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

Pluripotenz und Selbsterneuerung sind zwei definierenden Eigenschaften von embryonalen Stammzellen (ES-Zellen). Das Verständnis der zugrunde liegenden molekularen Mechanismus wird dadurch wesentlich erleichtert den Einsatz von ES-Zellen für Entwicklungsbiologie Studien, Krankheit Modellierung, der Wirkstoffforschung und in der regenerativen Medizin (Übersicht in 1,2). Um die Identifizierung und Charakterisierung neuer Regulatoren der ES-Zell-Wartungs-und Selbst-Erneuerung beschleunigen, entwickelten wir eine Fluoreszenz-Reporter-basierter Assay zur quantitativen Messung der Selbst-Erneuerung Status in Maus-ES-Zellen unter Verwendung des Oct4GiP Zellen 3. Die Oct4GiP Zellen exprimieren das grün fluoreszierende Protein (GFP) unter der Kontrolle des Oct4-Gen Promotor-Region 4,5. Oct4 ist für ES-Zell-Selbsterneuerung benötigt, und ist stark in ES-Zellen exprimiert und schnell herunter reguliert während der Differenzierung 6,7. Als ein Ergebnis korreliert GFP-Expression und Fluoreszenz in den Reporter-Zellen treumit dem ES-Zell-Identität 5 und Fluoreszenz-aktivierten Zellsortierung (FACS)-Analyse kann verwendet werden, die sorgfältige Überwachung der Selbst-Erneuerung Status der Zellen auf der Ebene der Einzelzelle 3,8 sein. Gekoppelt mit RNAi, kann die Oct4GiP Reporter Assay verwendet, um schnell zu identifizieren und zu untersuchen Regulatoren der ES-Zell-Wartungs-und Selbst-Erneuerung 3,8 sein. Im Vergleich zu anderen Methoden zur Bestimmung von Selbst-Erneuerung, ist es bequemer, einfühlsam, quantitative und von geringeren Kosten. Es kann in 96 durchgeführt werden - oder 384-Well-Platten für eine groß angelegte Studien wie High-Throughput-Screens oder genetische Analyse Epistasie. Schließlich wird unter Verwendung anderen Linie-spezifischen Reporters ES-Zelllinien, kann der Assay hier beschriebenen auch modifiziert werden, Determination während der ES-Zelldifferenzierung zu untersuchen.

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

Wir beschreiben einen Fluoreszenz-Reporter-Assay schnell zu identifizieren und zu charakterisieren, Gene, die Maus embryonalen Stammzellen Wartungs-und Selbst-Erneuerung zu regulieren.

Oct4GiP Reporter Assay auf Gene, die Maus embryonalen Stammzellen Wartungs-und Self-Study Erneuerung Regulate

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

Research

JoVE Journal

Biology

10.4K Aufrufe.  National Institute of Environmental Health Sciences. Wir beschreiben einen Fluoreszenz-Reporter-Assay schnell zu identifizieren und zu charakterisieren, Gene, die Maus embryonalen Stammzellen Wartungs-und Selbst-Erneuerung zu regulieren.

Serie: 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.

Ableitung von hämatopoetischen Stammzellen aus embryonalen Stammzellen der Maus

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 ...

Forschung

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.

Gewinnung menschlicher embryonaler Stammzellen durch 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 und in vivo Biolumineszenz Reporter Gene Imaging von humanen embryonalen Stammzellen

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.

Kultur und Wartung von humanen embryonalen Stammzellen

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.

Die Verwendung von SC1 (Pluripotin) zu MESC Self-Erneuerung in der Abwesenheit von LIF-Support

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.

Isolation und Ableitung von embryonalen Maus-Keimzellen

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-mount Immunhistochemische Analyse für Embryonale Limb Haut Gefäßsystem: ein Modellsystem zur Vascular Branching Morphogenese in Embryo-Studie

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.

Geändert Maus embryonalen Stammzellen basierte Assay zur Quantifizierung von Kardiogener Induction Efficiency

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.

Chromatin Immunopräzipitation-Assay für Tissue-spezifische Gene mit Frühzeitige muriner Embryonen

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.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

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