Agradecemos Brad Lackford para leitura e edição do manuscrito. Esta pesquisa foi financiada pelo Instituto Nacional de Ciências de Saúde Ambiental, National Institutes of Health Research Program intramuros Z01ES102745 (GH).

1. Oct4GiP mouse manutenção de células ES Células Oct4GiP foram gentilmente fornecido pelo Dr. Austin Smith. Eles foram derivados a partir dos ratinhos 129/Ola que transportam um transgene Oct4-GFPiresPac 4,5. Eles são mantidas em placas revestidas com gelatina de cultura de tecidos em meio ESGRO completa mais clonal grau (Millipore), ou em meio M15: DMEM (Invitrogen) suplementado com 15% de FBS, 1000 U / ml ESGRO (Millipore), 1x não- aminoácidos essenciais (Invitrogen), 1x EmbryoMax Nucleasides (Millipore), e 10 uM β-mercaptoetanol. <ol><li> Revestir placas com 0,1% de gelatina (Sigma) à temperatura ambiente durante 30 minutos (0,1 ml gelatina solução cm / 2). </li><li> Placa células Oct4GiP em gelatina-placas revestidas a ~ 2 x 10 4 cm / 2. </li><li> Dividir as células de dois em dois dias com tripsina a 0,05%. </li></ol> 2. Transfection siRNA em células Oct4GiP O ensaio de repórter Oct4GiP é mais convenienqüentemente realizado em gelatina-revestido fundo plano 96 - ou 384-bem placas (Corning ou BD) no meio de M15. O procedimento geral é descrito na Figura 1. <ol><li> siRNA lipídios montagem complexos: <ol><li> Para a transfecção em placas de 96 poços, montar siRNA-lipídicos complexos em U-inferior placas de 96 poços. </li><li> Em cada poço, misturar 10 uL OptiMEM (Invitrogen) e 0,3 ul Lipofectamine 2000 (Invitrogen) e incubar à temperatura ambiente durante 5 min. </li><li> Adicionar 5 pmol siRNA à mistura lípido-OptiMEM e incubar durante mais 15 minutos. O siRNA: razão de lípido é de 100 pmol: 6ul. </li><li> Transferir os complexos siRNA-lipídicos em OptiMEM a partir da placa de U-baixo para a gelatina revestido placa de fundo plano com uma pipeta multi-canal. </li><li> Para a transfecção em 384 poços, preparar os complexos siRNA-lipídicos utilizando Lipofectamine 0,1 uL de 2000 e 2 pmol siRNA em 10 OptiMEM uL. </li></ol></li></ol> Nota: A concentração final em siRNAas transfecções é de 50 nM. Realizar biológica 3-4 repetições para cada transfecção do siRNA. Configure misturas mestras dos complexos siRNA-lipídicos na placa de U-bottom e alíquota para o plano de fundo chapa de gelatina-revestido. <ol start="2"><li> Oct4GiP plaqueamento de células e de transfecção: <ol><li> Para a transfecção em placas de 96 poços, recolher as células Oct4GiP e ressuspender em meio M15-los fresco a 9 x 10 4 células / ml. </li><li> Adicionar 100 uL da suspensão de células para cada poço utilizando uma pipeta multi-canal. </li><li> Misturar a suspensão de células com os pré-alíquotas siRNA lípidos complexos no poço por pipetagem cima e para baixo 3-5 vezes. Alterar dicas para transfecções siRNA diferentes. </li><li> Para a transfecção em placas de 384 poços, as células Oct4GiP Ressuspenda a 6 x 10 4 células / ml e 30 aliquota de suspensão de células uL a cada poço. </li></ol></li></ol> Nota: A densidade de plaqueamento óptima de células é geralmente de 3 x 10 5 células / cm 2, mas may requerem otimização de siRNAs que afetam drasticamente o crescimento celular ou viabilidade. Densidade plaqueamento baixa irá conduzir a sobrevivência da célula pobre durante a transfecção. Densidade de plaqueamento alta levará a fundo elevado, devido à elevada diferenciação celular induzida confluência. Se necessário, a densidade de plaqueamento de ensaio entre 2-4 x 10 5 células / cm 2. <ol start="3"><li> Troca de meio: <ol><li> Remova o meio de idade, utilizando multi-canal de aspiração (Corning) ou vácuo varinha (VP-Científico). Ter cuidado para não riscar as células no fundo das placas. </li><li> Alimentar as células com meio fresco de células ES (100 uL para a placa de 96 poços e 30 uL de 384-bem placa) usando uma pipeta multi-canal a cada dia. </li></ol></li></ol> 3. Análise FACS <ol><li> Dissociação celular: <ol><li> Quatro dias após a transfecção, remover média usando multi-canal de aspiração (Corning) ou de vácuo varinha (VP-Scientific). </li><li> Para placas de 96 poços, lavar as células uma vez com 100μl de PBS. </li><li> Adicionar 25 ul de tripsina a 0,25% a cada poço utilizando uma pipeta multi-canal. </li><li> Incubar a temperatura ambiente durante 5 minutos com agitação ocasional, e inspeccione visualmente para assegurar separação completa das células. </li><li> Adicionar 90 ul de PBS com FBS 10% para inactivar a tripsina e dissociam-se as células em uma única suspensão celular por pipetagem repetida. </li><li> Para 384 poços, dissociar as células com 10 ul de tripsina e extingue-se com 30 ul de PBS com FBS 10%. </li></ol></li><li> Análise FACS: <ol><li> Analise a fluorescência da GFP no analisador FACS BD LSRII equipado com a unidade de HTS ou outro com o mesmo equipamento analisador FACS (tais como Accuri ou Intellicyt). </li><li> Utilizar o modo de alto rendimento no HTS e analisar 10 ul de suspensão de células. Definir o limite de contagem de 1,0 x 10 4 células. </li><li> Ajuste a tensão PMT e limiar para capturar as células na trama para frente contra o dispersor lateral. Porta para a população de células vivas no FORWard vs enredo lado dispersão. </li><li> Criar um histograma para o canal de GFP. Definir o portão no canal de modo que a GFP ~ 10% das células parecem ser GFP-negativa nas células simulados ou controlo siRNA-transfectadas. </li><li> Determinar% GFP-negativas células de cada tratamento: células diferenciadas% =% GFP-negativas células. Compare as células diferenciadas% entre experimentais e controle de siRNA-transfecções usando 2-tailed t-teste, e marcar as visitas positivas com valores de p &lt;0,01. </li></ol></li></ol> 4. Os resultados representativos Oct4, Nanog, e Sox2 são três genes que desempenham papéis cruciais na manutenção da célula ES auto-renovação 6,7,9-11. Figura 2 mostra que o ensaio de repórter Oct4GiP pode facilmente detectar a diferenciação causada por silenciar estes factores no células-tronco embrionárias Oct4GiP. A Figura 2A mostra as células ES são Oct4GiP GFP-positivo quando mantidos como ES<html> células. Figura 2B mostra o gráfico de dispersão para a frente vs lado e do histograma do canal de GFP das células Oct4GiP transfectadas com o controlo ou Oct4 de siRNAs. É necessário portão para células vivas no gráfico de dispersão para a frente vs lado, como as células mortas e detritos são GFP-negativa e irá aumentar de fundo. Por outro lado, dupleto discriminação para excluir aglomerados de células possíveis nem sempre é necessária. Nas células de controlo siRNA-transfectadas, a grande maioria das células deve ser GFP-positivo. Se óbvias GFP-negativas populações estão presentes nos poços de controlo de siRNA-transfectadas, as células Oct4GiP de partida ou o procedimento de transfecção pode ter sido comprometida eo resultado pode não ser interpretável. A Figura 2C mostra o gráfico de barras de células diferenciadas% (células diferenciadas% =% GFP-negativas células) do controle, Oct4-, NANOG-, e Sox2 siRNA-células transfectadas. <html> d/3987/3987fig1.jpg &quot;/&gt; Figura 1. Esboço do ensaio repórter Oct4GiP. <img alt="A Figura 2" src="/files/ftp_upload/3987/3987fig2.jpg" /> Figura 2. Oct4GiP ensaio repórter pode detectar diferenciação de células ES causada por Nanog, Oct4, ou silenciamento Sox2. A) E14Tg2a (de tipo selvagem de linha, preto) e Oct4GiP (linha verde), as células foram analisadas por FACS. Histograma da GFP-canal mostra que as células são Oct4GiP GFP-positivo. B) As células foram transfectadas Oct4GiP em placas de 96 poços com o controle ou Oct4 siRNA e FACS analisados ​​4 dias após a transfecção. As células para a frente vs parcelas laterais de dispersão e histogramas da GFP-canal das células transfectadas são mostrados. C) Oct4GiP foram transfectadas com o Controlo-,-Nanog, Oct4-, ou-Sox2 siRNA. As células diferenciadas% foi determinada a partir da% GFP-negativa células 4 dias após a transfecção, e foi desenhado como média + / - erro padrão da média (n = 4).f = &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot; target = &quot;_blank&quot;&gt; Clique aqui para ver maior figura.

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Não há conflitos de interesse declarados.

O ensaio repórter Oct4GiP descrevemos acima pode medir quantitativamente o grau de auto-renovação vs diferenciação. Em comparação com outros métodos disponíveis, tais como o 12 baseada na morfologia e proliferação / viabilidade baseado ensaios, oferece uma maior sensibilidade e rendimento, bem como uma medição mais directa do estado de células ES. É, portanto, bem adequado para grande escala telas e análise epistasia genética. Na verdade, nós e os outros têm utilizado com sucesso o ensaio r...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> ESGRO grau médio completo acrescido clonal </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (glucose elevada 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 (reduzir o meio de soro) </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 (Non-aminoácidos essenciais) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Nucleosídeos de 100x para a célula ES </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> 2-mercaptoetanol </td><td> Sigma </td><td> M7522-100ml </td></tr><tr><td> ES-qualificada de soro fetal bovino </td><td> Invitrogen </td><td> 10437 </td></tr><tr><td> SiRNA Nanog </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> SiRNA Sox2 </td><td> Dharmacon </td><td> M-058.489-01 </td></tr></tbody></table> SiRNA Controle: duplex siRNA alvo firefly luciferase (5&#39;-CGTACGCGGAATACTTCGA), sintetizado pelas Dharamcon.

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

Pluripotência e auto-renovação são duas características definidoras das células-tronco embrionárias (células ES). O entendimento do mecanismo molecular vai facilitar muito o uso de células-tronco embrionárias para estudos de biologia do desenvolvimento, modelagem de doenças, descoberta de drogas e da medicina regenerativa (revisto em 1,2). Para acelerar a identificação e caracterização de novos reguladores de manutenção de células ES e auto-renovação, foi desenvolvido um ensaio baseado em fluorescência repórter para medir quantitativamente o estado de auto-renovação em células ES de rato usando as células Oct4GiP 3. As células Oct4GiP expressar a proteína verde fluorescente (GFP), sob o controlo da região promotora do gene Oct4 4,5. Oct4 é necessária para a célula ES auto-renovação, e é altamente expressa em células ES e rapidamente para baixo-regulado durante a diferenciação 6,7. Como resultado, a GFP expressão e de fluorescência nas células repórter correlaciona fielmentecom a célula de identidade ES 5, e fluorescência-activated separação de células (FACS) análise pode ser usado para acompanhar de perto o estado de auto-renovação das células ao nível única célula 3,8. Juntamente com RNAi, o ensaio repórter Oct4GiP pode ser usada para rapidamente identificar e estudar os reguladores de manutenção de células ES e auto-renovação 3,8. Em comparação com outros métodos para ensaiar a auto-renovação, é mais conveniente, sensível, quantitativa, e de menor custo. Ela pode ser realizada em 96 - ou 384-bem placas para estudos em larga escala, tais como de alto rendimento telas ou de análise de epistasia genética. Finalmente, por meio de outros linhagem específicos repórter linhas de células ES, o ensaio descrevemos aqui também pode ser modificada para estudar especificação destino durante a diferenciação 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

Nós descrevemos um ensaio repórter de fluorescência para rapidamente identificar e caracterizar genes que regulam a manutenção do mouse células-tronco embrionárias e auto-renovação.

Oct4GiP Ensaio Reporter para o Estudo de genes que regulam o mouse Manutenção células-tronco embrionárias e auto-renovação

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 visualizações.  National Institute of Environmental Health Sciences. Nós descrevemos um ensaio repórter de fluorescência para rapidamente identificar e caracterizar genes que regulam a manutenção do mouse células-tronco embrionárias e auto-renovação.

Vídeo: 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.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

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 ...

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.

Derivação de células estaminais embrionárias 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 imagens Gene Reporter bioluminescência de células estaminais embrionárias 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.

Cultura e Manutenção de células estaminais embrionárias 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.

O uso de SC1 (Pluripotin) para Suporte MESC auto-renovação na ausência 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.

Isolamento e derivação de células embrionárias de rato 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-mount Análise imuno-histoquímica para Vascularização embrionárias Limb pele: um sistema modelo para estudar Morfogênese Branching Vascular em Embrião

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 embrionárias de camundongos com células-tronco Ensaio baseado para quantificar a eficiência de indução cardiogênico

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.

Ensaio para imunoprecipitação da cromatina Genes Tissue-específicos usando embriões em início de carreira do rato

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.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

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