Ringraziamo Brad Lackford per la lettura e la modifica del manoscritto. Questa ricerca è stata finanziata dal National Institute of Environmental Health Sciences, National Institutes of Health Research Programma intramurale Z01ES102745 (a GH).

1. Oct4GiP mouse cellule ES di manutenzione Cellule Oct4GiP sono stati gentilmente forniti dal Dr. Austin Smith. Essi sono stati ricavati dai topi portatori di una 129/Ola Oct4-GFPiresPac transgene 4,5. Essi sono mantenuti in piastre rivestite con tessuti gelatina coltura nella più completa clonale ESGRO grado medio (Millipore), o nel mezzo M15: DMEM (Invitrogen) supplementato con 15% FBS, 1000 U / ml ESGRO (Millipore), 1x non aminoacidi essenziali (Invitrogen), 1x Nucleasides EmbryoMax (Millipore), e 10 pM β-mercaptoetanolo. <ol><li> Ricoprire piastre con 0,1% di gelatina (Sigma) a temperatura ambiente per 30 minuti (0,1 ml soluzione di gelatina / cm 2). </li><li> Oct4GiP cellule a piastre rivestite di gelatina in piastre a ~ 2 x 10 4 / cm 2. </li><li> Dividere le celle ogni due giorni con tripsina 0,05%. </li></ol> 2. Transfection siRNA in cellule Oct4GiP Il saggio giornalista Oct4GiP è più convenienTLY effettuata in gelatina rivestita con fondo piatto 96 - o 384 pozzetti (Corning o BD) in mezzo M15. La procedura generale è illustrato nella figura 1. <ol><li> siRNA-lipidi complessi assembly: <ol><li> Per la trasfezione in piastre da 96 pozzetti, assemblare siRNA-lipidi complessi a U-bottom piastre a 96 pozzetti. </li><li> In ogni pozzetto, miscelare 10 microlitri OptiMEM (Invitrogen) e 0,3 pl Lipofectamine 2000 (Invitrogen) e incubare a temperatura ambiente per 5 min. </li><li> Aggiungere 5 pmol siRNA al lipide-OptiMEM miscela e incubare per altri 15 minuti. Il siRNA: rapporto di lipidi è 100 pmol: 6UL. </li><li> Trasferire i siRNA-lipidi complessi in OptiMEM dalla U-piastra inferiore alla gelatina-rivestito a fondo piatto piatto con una pipetta multicanale. </li><li> Per la trasfezione in piastre da 384 pozzetti, preparare i siRNA-lipidi complessi che utilizzano 0,1 microlitri Lipofectamine 2000 e del 2 siRNA pmol OptiMEM in 10 microlitri. </li></ol></li></ol> Nota: la concentrazione finale di siRNAle trasfezioni è di 50 nM. Effettuare biologica 3-4 repliche per ogni trasfezione siRNA. Impostare miscele master dei siRNA-lipidi complessi della U-piastra inferiore e aliquota nel fondo piatto rivestito di gelatina piatto. <ol start="2"><li> Oct4GiP placcatura cella e trasfezione: <ol><li> Per la trasfezione in piastre da 96 pozzetti, raccogliere le cellule Oct4GiP e risospendere in mezzo fresco M15 a 9 x 10 4 cellule / ml. </li><li> Aggiungere 100 pl della sospensione cellulare a ciascun pozzetto usando una pipetta multicanale. </li><li> Mescolare la sospensione cellulare con le pre-aliquotati siRNA di lipidi complessi nel bene Pipette su e giù per 3-5 volte. Modificare suggerimenti per trasfezioni diversi siRNA. </li><li> Per la trasfezione in piastre a 384 pozzetti, le cellule Oct4GiP risospendere a 6 x 10 4 cellule / ml e la sospensione cellulare aliquota 30 microlitri in ciascun pozzetto. </li></ol></li></ol> Nota: La densità ottimale placcatura cellulare è di solito 3 x 10 5 cellule / cm 2, ma may richiedono ulteriore ottimizzazione per i siRNA che drammaticamente influenzano la crescita delle cellule o la vitalità. Bassa densità di placcatura porterà alla sopravvivenza delle cellule poveri durante trasfezione. Alta densità di placcatura porterà a sfondo elevato a causa di elevata differenziazione confluenza cellulare indotta. Se necessario, placcatura densità prova tra 2-4 x 10 5 cellule / cm 2. <ol start="3"><li> Cambio medio: <ol><li> Rimuovere vecchio mezzo con multi-canale aspiratore (Corning) o il vuoto bacchetta (VP-scientifico). Fare attenzione a non graffiare le cellule nella parte inferiore delle piastre. </li><li> Alimentare le cellule con mezzo fresco cellule ES (100 microlitri per piastra a 96 pozzetti e 30 microlitri per piastra a 384 pozzetti) usando pipetta multicanale ogni giorno. </li></ol></li></ol> 3. FACS analisi <ol><li> Dissociazione cellulare: <ol><li> Quattro giorni dopo la transfezione, rimuovere media multi-canale con aspiratore (Corning) o il vuoto bacchetta (VP-scientifico). </li><li> Per piastre a 96 pozzetti, lavare una volta con cellule 100μl PBS. </li><li> Aggiungere 25 microlitri tripsina 0,25% ad ogni pozzetto utilizzando una pipetta multicanale. </li><li> Incubare a temperatura ambiente per 5 minuti con agitazione occasionale, e ispezionare visivamente per garantire il completo distacco delle cellule. </li><li> Aggiungere 90 microlitri di PBS con 10% FBS per inattivare la tripsina e dissociare cellule in una sospensione singola cella pipettando ripetuta. </li><li> Per piastre a 384 pozzetti, dissociare le cellule con 10 microlitri tripsina e quench con 30 pl PBS con 10% FBS. </li></ol></li><li> FACS analisi: <ol><li> Analizzare la fluorescenza GFP sull&#39;analizzatore BD LSRII FACS dotata dell&#39;unità HTS o altro analizzatore analogamente dotato FACS (come Accuri o Intellicyt). </li><li> Utilizzare la modalità high-throughput sul HTS e analizzare 10 microlitri di sospensione cellulare. Impostare la soglia conteggio a 1.0 x 10 4 cellule. </li><li> Regolare la tensione di PMT e la soglia di catturare le cellule nella trama in avanti rispetto al side-scatter. Porta per la popolazione di cellule dal vivo nel DeviazioneARD vs side-scatter plot. </li><li> Creare un grafico a istogramma per il canale GFP. Impostare la porta nel canale GFP in modo che circa il 10% delle cellule risultano GFP-negativi nelle cellule di controllo o finti-siRNA transfettate. </li><li> Determinare sulla GFP-negativi cellule provenienti da ogni trattamento: le cellule differenziate sulla =% GFP-negativi cellule. Confronta le cellule sulla differenziate tra sperimentali-e control-siRNA trasfezioni con due code t-test, e segnare i successi positivi con valori di p &lt;0.01. </li></ol></li></ol> 4. Risultati rappresentativi Oct4, Nanog, Sox2 e sono tre geni che giocano un ruolo critico nel mantenimento delle cellule ES auto-rinnovamento 6,7,9-11. La figura 2 mostra che il saggio giornalista Oct4GiP può facilmente rilevare la differenziazione causato da mettere a tacere questi fattori nel Oct4GiP cellule ES. La figura 2A mostra le cellule staminali embrionali sono Oct4GiP GFP-positive quando mantenuti ES<html> cellule. Figura 2B mostra la dispersione in avanti contro lato e l&#39;istogramma del canale GFP delle cellule Oct4GiP trasfettate con il controllo o Oct4 di siRNA. E &#39;necessario cancello per le cellule vive nel grafico a dispersione in avanti rispetto a lato, come le cellule morte e detriti sono GFP-negativi e aumenterà sfondo. D&#39;altra parte, doppietto discriminazione di escludere possibili grumi di cellule non è sempre necessaria. Nelle controllo siRNA cellule trasfettate, la stragrande maggioranza delle cellule deve essere GFP-positive. Se ovvie GFP-negativi popolazioni sono presenti nei pozzi di controllo siRNA trasfettate, le cellule Oct4GiP di partenza o la procedura di trasfezione può essere stata compromessa e il risultato non può essere interpretabile. La figura 2C mostra il grafico a barre delle cellule sulla differenziate (cellule differenziate sulla =% cellule GFP-negativi) dal controllo-,-Oct4, Nanog-e Sox2-siRNA cellule trasfettate. <html> d/3987/3987fig1.jpg &quot;/&gt; Figura 1. Schema del saggio giornalista Oct4GiP. <img alt="Figura 2" src="/files/ftp_upload/3987/3987fig2.jpg" /> Figura 2. Saggio giornalista Oct4GiP in grado di rilevare la differenziazione delle cellule ES causato da Nanog, Oct4, Sox2 e silenziamento. A) E14Tg2a (wild-type, linea nera) e Oct4GiP (linea verde), le cellule sono stati analizzati mediante FACS. Istogramma della GFP-canale dimostra che le cellule sono Oct4GiP GFP-positivi. B) Oct4GiP cellule sono state trasfettate in piastre da 96 pozzetti con il controllo-o-Oct4 siRNA e FACS analizzati 4 giorni dopo la trasfezione. Forward vs grafici a dispersione laterali e istogrammi della GFP-canale delle cellule trasfettate sono mostrate. C) Oct4GiP cellule sono state trasfettate con il controllo-,-Nanog, Oct4-, o-Sox2 siRNA. Le celle sulla dissociati è stata determinata dalla% GFP-negativo celle 4 giorni dopo la transfezione, ed è stata tracciata come media + / - errore standard della media (n = 4).f = target &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot; = &quot;_blank&quot;&gt; Clicca qui per ingrandire la figura.

<ol>
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Il saggio giornalista Oct4GiP abbiamo descritto sopra può misurare quantitativamente il grado di auto-rinnovamento vs differenziazione. Rispetto ad altri metodi disponibili, come la morfologia a base 12 e la proliferazione / vitalità saggi basati, offre maggiore sensibilità e velocità, nonché una misura più diretta dello stato della cellula ES. E &#39;quindi adatto per i grandi schermi e analisi epistasi genetica. In effetti, noi e altri hanno utilizzato con successo il saggio giornalista Oct4GiP per la...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> ESGRO medio grado completa e clonale </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (High glucosio 1X) </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> 0,25% tripsina-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 (riduzione media del siero) </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 (aminoacidi non essenziali) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Nucleosidi 100x di cellule ES </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> 2-mercaptoetanolo </td><td> Sigma </td><td> M7522-100ml </td></tr><tr><td> ES-qualificato siero fetale bovino </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> SiRNA di controllo: duplex siRNA mira lucciola luciferasi (5&#39;-CGTACGCGGAATACTTCGA) sintetizzato Dharamcon.

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

Pluripotenza e di auto-rinnovamento sono due caratteristiche che definiscono le cellule staminali embrionali (cellule ES). Comprendere il meccanismo molecolare agevolerà notevolmente l&#39;uso di cellule staminali embrionali per gli studi di biologia dello sviluppo, modelli di malattia, scoperta di farmaci, e la medicina rigenerativa (recensione in 1,2). Per velocizzare l&#39;identificazione e la caratterizzazione di nuovi regolatori di manutenzione delle cellule ES e di auto-rinnovamento, abbiamo sviluppato una fluorescenza giornalista-based test per misurare quantitativamente il self-renewal stato in cellule staminali embrionali di topo usando le cellule Oct4GiP 3. Le cellule Oct4GiP esprimono la proteina fluorescente verde (GFP) sotto il controllo della regione del promotore del gene Oct4 4,5. Oct4 è necessaria per cellule ES auto-rinnovamento, ed è altamente espressa nelle cellule ES e rapidamente down-regolato durante la differenziazione 6,7. Come risultato, l&#39;espressione della GFP e fluorescenza nelle cellule del reporter correla fedelmentecon l&#39;identità della cellula ES 5, e fluorescenza selezione delle cellule attivate (FACS) analisi può essere utilizzato per monitorare attentamente la auto-rinnovamento stato delle celle a livello di singola cellula 3,8. Accoppiato con RNAi, il saggio giornalista Oct4GiP può essere utilizzato per identificare rapidamente e studiare regolatori di manutenzione cellule ES e auto-rinnovamento 3,8. Rispetto ad altri metodi per saggiare auto-rinnovamento, è più conveniente, sensibile, quantitativa, e di costo inferiore. Essa può essere effettuata in 96 - o 384 pozzetti per studi su larga scala, quali ad alto rendimento schermi o analisi epistasi genetica. Infine, utilizzando altri lineage-specifici reporter di linee cellulari ES, il saggio qui descritte possono anche essere modificato per studiare il destino specifica durante la differenziazione delle cellule 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

Descriviamo un saggio di fluorescenza giornalista per identificare rapidamente e caratterizzare i geni che regolano il mouse sulle cellule staminali embrionali di manutenzione e di auto-rinnovamento.

Oct4GiP Reporter Assay per studiare i geni che regolano il mouse sulle cellule staminali embrionali di manutenzione e di auto-rinnovamento

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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JoVE Journal

Biology

10.4K Views.  National Institute of Environmental Health Sciences. Descriviamo un saggio di fluorescenza giornalista per identificare rapidamente e caratterizzare i geni che regolano il mouse sulle cellule staminali embrionali di manutenzione e di auto-rinnovamento.

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.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

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 ...

Ricerca

Biologia

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.

Derivazione di cellule staminali embrionali umane da 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 e in Imaging bioluminescenza gene reporter in vivo di cellule staminali embrionali umane

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.

Cultura e manutenzione di cellule staminali embrionali umane

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;uso di SC1 (Pluripotin) a supporto Mesc auto-rinnovamento in assenza di 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;isolamento e la derivazione di cellule embrionali del mouse Germinal

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.

Con tutto il supporto per l&#39;analisi immunoistochimica vasi embrionali pelle degli arti: un sistema modello per studiare morfogenesi vascolare diramazione in embrione

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.

Modificato il mouse sulle cellule staminali Saggio base per quantificare l&#39;efficienza di induzione cardiogeno

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.

Saggio di immunoprecipitazione della cromatina per geni tessuto-specifici utilizzando nella fase iniziale di embrioni di topo

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.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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