Damos las gracias a Brad Lackford para la lectura y edición del manuscrito. Esta investigación fue apoyada por el Instituto Nacional de Ciencias de Salud Ambiental, Institutos Nacionales de Salud, Programa de Investigación Intramural Z01ES102745 (GH).

1. Oct4GiP madre embrionarias de ratón de mantenimiento de la célula Oct4GiP células fueron amablemente proporcionados por el Dr. Austin Smith. Se deriva de los ratones portadores 129/Ola un transgén Oct4-GFPiresPac 4,5. Ellos se mantienen en gelatina placas recubiertas de cultivo de tejidos en el medio ESGRO grado completa más clonal (Millipore), o en el medio M15: DMEM (Invitrogen) suplementado con FBS al 15%, 1000 U / ml ESGRO (Millipore), 1x para no- aminoácidos esenciales (Invitrogen), 1x Nucleasides EmbryoMax (Millipore), y 10 mM mercaptoetanol β-. <ol><li> Coat con placas% de gelatina 0,1 (Sigma) a temperatura ambiente durante 30 minutos (0,1 ml de solución de gelatina / cm 2). </li><li> Células de la placa Oct4GiP en gelatina revestida de placas de 2 x 10 ~ 4 / cm 2. </li><li> Dividir las celdas de cada dos días con 0,05% de tripsina. </li></ol> 2. siRNA transfección en células Oct4GiP El ensayo reportero Oct4GiP es más convenientemente lleva a cabo en gelatina recubierto con fondo plano 96 - o placas 384-pocillos (Corning o BD) en el medio M15. El procedimiento general se esboza en la Figura 1. <ol><li> siRNA-lípidos complejos de montaje: <ol><li> Para la transfección en placas de 96 pocillos, ensamble de lípidos complejos de siRNA en fondo en U placas de 96 pocillos. </li><li> En cada pocillo, mezclar 10 l OptiMEM (Invitrogen) y 0,3 l Lipofectamine 2000 (Invitrogen) y se incuba a temperatura ambiente durante 5 min. </li><li> Añadir 5 pmol ARNsi a la mezcla de lípidos OptiMEM e incubar durante otros 15 minutos. El siRNA: relación de los lípidos es de 100 pmol: 6ul. </li><li> Transferir los complejos de lípidos siRNA en OptiMEM de la placa de fondo en U a la revestida con gelatina de fondo plano placa con una pipeta de canales múltiples. </li><li> Para la transfección en placas de 384 pocillos, preparar los complejos de lípidos siRNA con 0,1 l Lipofectamine 2000 y 2 siRNA pmol en 10 OptiMEM l. </li></ol></li></ol> Nota: La concentración final de siRNAlas transfecciones es 50 nM. Llevar a cabo biológica 3-4 repeticiones para cada transfección de siRNA. Establecer las mezclas maestras de los complejos de lípidos siRNA en la placa de fondo en U y la alícuota en el fondo plano de gelatina recubierto de placas. <ol start="2"><li> Oct4GiP chapado de células y transfección: <ol><li> Para la transfección en placas de 96 pocillos, recoger las células Oct4GiP y resuspender en medio de frescas M15 a 9 x 10 4 células / ml. </li><li> Añadir 100 l de la suspensión de células en cada pocillo utilizando una pipeta de canales múltiples. </li><li> Mezclar la suspensión de células con los pre-alícuotas lípidos siRNA-complejos en el pozo por pipetear arriba y hacia abajo 3-5 veces. Cambiar las puntas de siRNA transfections diferentes. </li><li> Para la transfección en placas de 384-pozos, Resuspender las células Oct4GiP a 6 x 10 4 células / ml y 30 alícuota suspensión celular l a cada pocillo. </li></ol></li></ol> Nota: La densidad de célula de recubrimiento óptimo es generalmente de 3 x 10 5 células / cm 2, pero may requieren una mayor optimización de siRNAs que afectan drásticamente el crecimiento de células o la viabilidad. Baja densidad de placas dará lugar a la supervivencia celular deficiente durante la transfección. Densidad de placas de alta dará lugar a un fondo elevado debido a la diferenciación de alta confluencia celular inducida. Si es necesario, prueba de densidad de placas entre 2-4 x 10 5 células / cm 2. <ol start="3"><li> Cambio de medio: <ol><li> Eliminar el medio de edad con múltiples canales de aspiración (Corning) o el vacío varita (VP-científico). Tenga cuidado de no dañar las células en la parte inferior de las placas. </li><li> Alimentar a las células con un nuevo medio de células ES (100 l para la placa de 96 pozos y 30 l de 384 y placa) con pipeta multicanal todos los días. </li></ol></li></ol> 3. Análisis FACS <ol><li> Disociación celular: <ol><li> Cuatro días después de la transfección, eliminar el medio usando multi-canal de aspiración (Corning) o el vacío varita (VP-científico). </li><li> Para placas de 96 pocillos, lavar las células una vez con 100μl de PBS. </li><li> Añadir 25 l de tripsina 0,25% a cada pocillo utilizando una pipeta multicanal. </li><li> Incubar a temperatura ambiente durante 5 minutos con agitación ocasional, y una inspección visual para asegurar desprendimiento completo de las células. </li><li> Añadir 90 l de PBS con FBS al 10% para inactivar la tripsina y disociar las células en una suspensión de células individuales por pipeteado repetido. </li><li> Para placas de 384 pocillos, disociar las células con 10 l de tripsina y enfriamiento con 30 ul PBS con 10% de FBS. </li></ol></li><li> FACS análisis: <ol><li> Analizar la fluorescencia de GFP en el analizador BD LSRII FACS equipado con la unidad HTS u otro similar equipado analizador FACS (como Accuri o Intellicyt). </li><li> Utilice el modo de alto rendimiento en el HTS y analizar los 10 l de suspensión celular. Establecer el umbral de recuento de 1,0 x 10 4 células. </li><li> Ajuste la tensión de la PMT y el umbral para capturar las células en la trama hacia adelante frente a dispersión lateral. Puerta de la población de células vivas en la dwnARD vs lado gráfico de dispersión. </li><li> Crear un histograma para el canal de las buenas prácticas agrarias. Ajuste de la puerta en el canal de GFP de modo que ~ 10% de las células parecen ser GFP-negativo en las células transfectadas simuladas o control siRNA. </li><li> Determinar% negativas GFP-células de cada tratamiento: las células diferenciadas% =% de células GFP-negativos. Comparar las células diferenciadas% entre experimentales y de control de siRNA-transfecciones con dos colas t-test, y anotar los golpes positivos con los valores de p &lt;0.01. </li></ol></li></ol> 4. Los resultados representativos Oct4, Nanog, y Sox2 son tres genes que desempeñan papeles cruciales en el mantenimiento de células ES de auto-renovación 6,7,9-11. Figura 2 muestra que el reportero de ensayo Oct4GiP puede detectar fácilmente la diferenciación causada por el silenciamiento de estos factores en el Oct4GiP células madre embrionarias. La figura 2A muestra las células ES son Oct4GiP GFP-positivo cuando se mantiene como ES<html> células. Figura 2B muestra el diagrama de dispersión hacia adelante contra lado y el histograma del canal GFP de las células transfectadas con el Oct4GiP control o Oct4 siRNAs. Es necesario puerta para las células vivas en el gráfico de dispersión hacia adelante contra lado, como las células muertas y los desechos son GFP-negativo y aumentará fondo. Por otro lado, doblete discriminación para excluir posibles grumos de células no es siempre necesaria. En las células de control siRNA-transfectadas, la gran mayoría de las células debe ser GFP-positivo. Si evidentes las buenas prácticas agrarias-negativos poblaciones están presentes en los pozos de control de siRNA-transfectadas, las células Oct4GiP de partida o el procedimiento de transfección puede estar comprometida y el resultado no puede ser interpretable. La figura 2C muestra el gráfico de barras de las células diferenciadas% (células diferenciadas% =% GFP-negativos células) de control, Oct4, Nanog, y Sox2 siRNA-transfectadas las células. <html> d/3987/3987fig1.jpg &quot;/&gt; Figura 1. Esquema del reportero de ensayo Oct4GiP. <img alt="Figura 2" src="/files/ftp_upload/3987/3987fig2.jpg" /> Figura 2. Ensayo de Oct4GiP reportero puede detectar la diferenciación de células ES causada por Nanog, Oct4, Sox2 o el silenciamiento. A) E14Tg2a (de tipo salvaje, la línea de negro) y las células Oct4GiP (línea verde) se analizaron por FACS. Histograma de la GFP canales muestra que las células son las buenas prácticas agrarias Oct4GiP positivo. B) Las células fueron transfectadas Oct4GiP en placas de 96 pocillos con el control o Oct4 siRNA y FACS analizaron 4 días después de la transfección. Adelante vs gráficos de dispersión y los histogramas laterales de la GFP-canal de las células transfectadas se muestran. C) Las células Oct4GiP fueron transfectadas con el control, Nanog, Oct4, Sox2, o siRNA. Las células diferenciadas% se determinó a partir de la GFP% negativo células 4 días después de la transfección, y se representan como media + / - error estándar de la media (n = 4).f = &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot; target = &quot;_blank&quot;&gt; Haga clic aquí para ver más grande figura.

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No hay conflictos de interés declarado.

El reportero de ensayo Oct4GiP que describimos arriba cuantitativamente se puede medir el grado de auto-renovación frente a la diferenciación. En comparación con otros métodos disponibles, tales como la morfología 12 basada en la proliferación y / ensayos de viabilidad basado, que ofrece una mayor sensibilidad y el rendimiento, así como una medición más directa del estado de células ES. Por tanto, se adapta bien a gran escala pantallas y análisis epistasis genética. De hecho, nosotros y otros han ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> ESGRO de grado medio, y además de clonal </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (glucosa alta 1X) </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> 0,25% de 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 (reducir medio de suero) </td><td> Invitrogen </td><td> 31985 </td></tr><tr><td> ESGRO primer fondo (10 7 units/1ml) </td><td> Millipore </td><td> DAM1776540 </td></tr><tr><td> MEM AANE (no aminoácidos esenciales) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Nucleósidos 100X para células ES </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> 2-mercaptoetanol </td><td> Sigma </td><td> M7522-100 ml </td></tr><tr><td> ES-calificado de suero fetal 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-046256-02 </td></tr><tr><td> Sox2 siRNA </td><td> Dharmacon </td><td> M-058.489-01 </td></tr></tbody></table> El control de siRNA: dúplex siRNA dirigidos a la luciferasa de luciérnaga (5&#39;-CGTACGCGGAATACTTCGA) sintetizados por Dharamcon.

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

Pluripotencia y auto-renovación son dos características que definen a las células madre embrionarias (células ES). Entender el mecanismo molecular subyacente facilitará en gran medida el uso de células madre embrionarias para los estudios de biología del desarrollo, modelos de enfermedades, descubrimiento de fármacos y Medicina Regenerativa (revisado en 1,2). Para agilizar la identificación y caracterización de nuevos reguladores de mantenimiento de células ES y auto-renovación, hemos desarrollado un reportero de fluorescencia de base de ensayo para medir cuantitativamente el estado de auto-renovación en células madre embrionarias de ratón usando las células Oct4GiP 3. Las células Oct4GiP expresar la proteína verde fluorescente (GFP) bajo el control de la región promotora del gen Oct4 4,5. Oct4 se requiere para células ES de auto-renovación, y es altamente expresado en células madre embrionarias y rápidamente regulado a la baja durante la diferenciación 6,7. Como resultado, la expresión de GFP y fluorescencia en las células informadoras se correlaciona fielmentecon la identidad de células ES 5, y activado por fluorescencia de células de clasificación (FACS) el análisis se puede utilizar para controlar estrechamente el estado de auto-renovación de las células en el nivel de células individuales 3,8. Junto con ARNi, el ensayo reportero Oct4GiP se puede utilizar para identificar rápidamente y estudiar los reguladores de mantenimiento de células ES y autorrenovación 3,8. En comparación con otros métodos para el ensayo de auto-renovación, es más conveniente, sensible, cuantitativa, y de menor coste. Puede llevarse a cabo en 96 - o placas de 384-bien para estudios a gran escala, tales como pantallas de alto rendimiento o de análisis de epistasis genética. Finalmente, mediante el uso de otros linaje específico reportero líneas de células ES, el ensayo se describe aquí también se puede modificar para estudiar especificación destino durante la diferenciación de células 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

Se describe un reportero de ensayo de fluorescencia para identificar rápidamente y caracterizar los genes que regulan el mantenimiento de embriones de ratón con células madre y auto-renovación.

Oct4GiP reportero de ensayo para estudiar los genes que regulan la de embriones de ratón con células madre de mantenimiento y auto-renovación

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 vistas.  National Institute of Environmental Health Sciences. Se describe un reportero de ensayo de fluorescencia para identificar rápidamente y caracterizar los genes que regulan el mantenimiento de embriones de ratón con células madre y auto-renovación.

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.

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

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 ...

Investigación

Biología

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.

Derivación de células estaminales embrionarias humanas por 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 vivo de imágenes de bioluminiscencia gen reportero de células estaminales embrionarias humanas

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.

La cultura y el mantenimiento de células estaminales embrionarias humanas

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.

El uso de SC1 (Pluripotin) para apoyar MESC auto-renovación en la ausencia 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.

El aislamiento y la derivación de células embrionarias de ratón 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.

Todo el montaje El análisis inmunohistoquímico de la piel del miembro Vasculatura embrionarias: un sistema modelo para estudiar la morfogénesis vascular ramificación en embriones

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.

Modificado madre embrionarias de ratón basado en células para la cuantificación de la eficiencia de inducción cardiogénico

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.

Ensayo de inmunoprecipitación de la cromatina de los genes específicos de tejido utilizando etapa temprana de embriones de ratón

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.

Generación de ratones derivados de células madre pluripotentes inducidas

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