Los autores desean agradecer a Irene Choi para ilustrar las cabezas de embriones en la figura 1 y los otros miembros del laboratorio en busca de ayuda y discusión. Este trabajo es apoyado por el NIH subvención DE012728 (TW)

1. Preparar Dispasa II <ol><li> Preparar fresco Hepes-solución salina tamponada (HBS) (50 mM HEPES / KOH pH 7,4, 150 mM NaCl). Añadir 2 ml de 0,5 M Hepes / KOH pH 7,4 y 1,2 ml de NaCl 2,5 M en 16,8 ml ultrapura H 2 O. </li><li> Hacer 10 mg / ml Dispasa II. Pesar 0,2 g Dispase II (Roche, Cat # 4942078001) y disolver en 20 ml de HBS. Preparar alícuotas de 1,0 ml en tubos individuales de Eppendorf y se almacena a -20 ° C para almacenamiento a largo plazo. </li><li> Diluir 10 mg / ml Dispasa II 1:10 en PBS antes de su uso. Ponga el diluido II Dispasa sobre hielo. </li></ol> 2. Disecciona CD-1 a partir de embriones E10.5 Mujer Embarazada Todos los experimentos con animales se realizaron de acuerdo con los protocolos aprobados por el Cuidado de Animales de la Universidad de Colorado en Denver (UCD) y el Comité de uso. Ratones CD-1 (ICR) se obtuvieron de Harlan Laboratories (Indianapolis, IN). <ol><li> Limpiar la zona de disección y herramientas con 70% de etanol. </li><li> Eutanasiar embarazada CD-1 hembra con Aprobacióned protocolos. </li><li> Rocíe el abdomen con un 70% de etanol y luego abrir el abdomen. Use un juego de pinzas para tirar del útero y otro para arrancar el mesometrio del útero. Extirpar el útero del cuerpo. Repita el procedimiento para cuerno uterino en el otro lado del abdomen. </li><li> Brevemente enjuague el útero con PBS enfriado con hielo en un plato de Petri 10-cm para limpiarla de sangre. </li><li> Transferir el útero en una nueva placa de Petri con PBS enfriado con hielo. Separado en fragmentos de embriones de una sola cortando entre sitios de implantación. </li><li> Transferir un embrión en PBS helado bajo un microscopio estereoscópico. Use unas pinzas finas para arrancar la pared muscular del útero a partir de uno de los sitios de corte para exponer el embrión con el saco vitelino. Retire el saco vitelino y el amnios. La transferencia de embriones disecados a un plato de Petri de 6 cm con PBS en hielo. Continúe hasta que todos los embriones han sido recogidos de esta manera. </li></ol> 3. Dispasa II El tratamiento del <ol><li> Lavar todos los embriones con PBS en una placa de Petri de 6 cm. ReemplazarPBS con 10 ml diluido en PBS Dispase II. Cubrir la placa de Petri y se incuba a 37 ° C durante 25 minutos - una infección bacteriana incubadora o sala caliente debe ser suficiente - no hemos utilizado un CO 2 incubadora de cultivo de tejidos para este paso. </li><li> Tomar la placa de Petri a cabo y observar el embrión en un estereomicroscopio. El ectodermo se han aflojado, caracterizado por una clara diferencia entre esta capa de tejido y el mesénquima subyacente. Si no, incubar durante 5 min a 37 ° C. A continuación, coloque la placa de Petri en hielo. </li></ol> 4. Diseccionar las prominencias faciales intactas <ol><li> Utilice una pipeta de vidrio desechable (VWR, Cat # 14672-380) para transferir un Dispase II tratados embrión en PBS enfriado en hielo en una placa de Petri de 6 cm en un estereomicroscopio. </li><li> Utilice un par de pinzas para sujetar el embrión. El uso de otro par de pinzas finas para cortar cuidadosamente los límites de las prominencias faciales unidos a la cabeza (Figura 1 muestra los límites delas prominencias faciales). Iniciar desde un lado de la MnP, entonces la MxP, y FNP la última. Hacer lo mismo para el otro lado de diseccionar las prominencias faciales fuera intacto (Figura 2A). </li></ol> 5. Ectodermo facial independiente de mesénquima <ol><li> Tenga en cuenta las prominencias faciales bajo un microscopio estereoscópico con una distancia de trabajo de 3x. Utilice una pipeta Pasteur de vidrio larga para pipetear suavemente las prominencias faciales arriba y abajo 5 veces. Después de esto, el ectodermo es fácil de pelar. </li><li> Use un par de pinzas para sujetar las prominencias faciales. Utilice el otro par de pinzas para pelar el ectodermo muy lentamente. Figura 2B muestra las hojas separadas del ectodermo facial. </li><li> Utilice una nueva pipeta Pasteur de vidrio larga para transferir el ectodermo facial en un tubo de 1,5 ml Eppendorf con PBS en hielo. Transferir el mesénquima a otro tubo de 1,5 ml Eppendorf con PBS en hielo. </li><li> Utilice el nuevo hielo frío PBS para cada disección. Recoger muestras. </li><li> Lave tél las muestras con PBS. Se centrifuga a 4 ° C, 500 xg durante 3 min. </li><li> Aspirar el PBS utilizando pipetas Pasteur de vidrio largas. Si las muestras se van a utilizar para el chip de ensayo, vaya al paso 6.1. Para la extracción de RNA, vaya al paso 6.2. Para la extracción de proteínas, vaya al paso 6.3. </li></ol> 6. Las muestras de proceso para nuevos experimentos <ol><li> Las muestras para ensayo ChIP <ol><li> Añadir 1 ml de PBS con 1% de formaldehído para reticular. Homogeneizar las muestras. </li><li> Incubar a temperatura ambiente durante 10 min con rotación. </li><li> Añadir 110 l de 1,25 M de glicina a cada tubo para saciar formaldehído sin reaccionar. </li><li> Mezclar e incubar a temperatura ambiente durante 5 min. </li><li> Spin a 500 xg, a 4 ° C durante 3 min. Eliminar el sobrenadante. </li><li> Añadir 1 ml de PBS frío para lavar los tejidos. </li><li> Spin a 500 xg, a 4 ° C durante 3 min. </li><li> Retire PBS PBS y repetir lavar una vez. </li><li> Remover el sobrenadante, Snap congelar los tejidos en nitrógeno líquido. Guarde la samples a -80 ° C para la puesta en común posterior y el procesamiento. </li></ol></li><li> Las muestras para la extracción de ARN <ol><li> Coloque el ectodermo y el mesénquima facial en tubos que contienen RNAlater (Ambion). </li><li> Se incuban las muestras en solución RNAlater durante la noche a 4 ° C para permitir la penetración profunda del tejido. </li><li> Almacenar a -20 ° C para la puesta en común posterior y procesamiento. </li></ol></li><li> Las muestras para la extracción de proteínas </li></ol> Añadir tampón de lisis a la muestra para su posterior procesamiento. O SNAP congelar los tejidos en nitrógeno líquido. Almacenar las muestras a -80 º C para la puesta en común posterior y procesamiento. Notas <ol><li> Las limitaciones de tiempo. Planee en 4-6 horas desde el inicio de la disección hasta el comienzo de la sección 6. </li><li> Cuando competente, toma ~ 10-15 min por embrión para realizar las secciones 4 y 5 (la prominencia disección y separación ectodermo). </li><li> Si las muestras son para el aislamiento de ARN, cada solution debe ser tratada con pirocarbonato de dietilo primero. También limpie las pinzas y el área de trabajo con RNasa Away. </li><li> Mantener las muestras en hielo en todo momento, excepto para el tratamiento Dispasa II y disecciones. Cambie al nuevo PBS enfriado con hielo para cada disección para asegurarse de que el sistema de cartilla utilizada para la disección en frío. </li><li> Pinzas cortantes son críticos para obtener disección precisa. Limpie la pinza completamente para evitar cualquier contaminación. </li><li> Para recoger muestras de diferentes prominencias, diseccionar cada prominencia por separado después de Dispasa tratamiento II. A continuación, separar el ectodermo y el mesénquima. </li><li> Después del tratamiento con Dispasa II, el ectodermo facial es fácil de pelar. Preste especial atención a la contaminación del ectodermo otro. Deseche todos los tejidos potencialmente contaminados. </li><li> Una vez que comience, no te detengas hasta que las muestras alcancen el punto apropiado en la Sección 6. Finalizar separación del ectodermo facial y mesénquima para todos los embriones lo más rápido que puedas. </li><li> El correoactividades nzyme de Dispasa II podría ser diferente entre lotes y marcas. Es necesario ajustar el tiempo de tratamiento para diferentes marcas y los lotes de Dispase II. </li></ol>

<ol>
	<li>Wilkie, A. O., Morriss-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+craniofacial+development+and+malformation.">Genetics of craniofacial development and malformation.</a> Nat. Rev. Genet. 2, 458-468 (2001).</li><li>Gritli-Linde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2+-+The+Etiopathogenesis+of+cleft+lip+and+cleft+palate:+usefulness+and+caveats+of+mouse+models.">Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.</a> Current Topics in Developmental Biology. 84, 37-138 (2008).</li><li>Schutte, B. C., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+and+factors+of+orofacial+clefts.">The many faces and factors of orofacial clefts.</a> Human Molecular Genetics. 8, 1-7 (1999).</li><li>Chai, Y., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+craniofacial+morphogenesis.">Recent advances in craniofacial morphogenesis.</a> Dev. Dyn. 235, 2353-2375 (2006).</li><li>Jiang, R., Bush, J. O., Lidral, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+upper+lip:+Morphogenetic+and+molecular+mechanisms.">Development of the upper lip: Morphogenetic and molecular mechanisms.</a> Dev. Dyn. 235, 1152-1166 (2006).</li><li>Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectodermal+WNT/&#946;-catenin+signaling+shapes+the+mouse+face.">Ectodermal WNT/&#946;-catenin signaling shapes the mouse face.</a> Developmental Biology. 349, 261-269 (2011).</li><li>Feng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+analysis+of+gene+expression+during+growth+and+fusion+of+the+mouse+facial+prominences.">Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.</a> PLoS ONE. 4, e8066 (2009).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat. Genet. 37 (11), 1181-1186 (2005).</li><li>Nagai, T., Aruga, J., Takada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+the+mouse+Zic1,+Zic2,+and+Zic3+gene+suggests+an+essential+role+for+Zic+genes+in+body+pattern+formation.">The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.</a> Developmental Biology. 182, 299-313 (1997).</li><li>Britsch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+Sox10+is+a+key+regulator+of+peripheral+glial+development.">The transcription factor Sox10 is a key regulator of peripheral glial development.</a> Genes Dev. 15 (1), 66-78 (2001).</li></ol>

Los autores no tienen intereses en conflicto o los conflictos asociados con el contenido de este artículo.

Este protocolo proporciona un método sencillo para separar ectodermo embrionario del ratón facial y el mesénquima basado en una etapa de tratamiento inicial Dispase II. En estudios anteriores, hemos realizado la disección de las prominencias faciales antes del tratamiento Dispasa II, pero hemos encontrado consistentemente que el mesénquima se vuelve &quot;pegajoso&quot; y más difícil de manipular. Nuestro nuevo protocolo evita este problema. Combinación Dispase II tratamiento con la fuerza física suave pipetean...

<table>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <td>Dispase II (neutral protease, grade II)</td>
 <td>Roche</td>
 <td>4942078001</td>
 <td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &deg;C . </td>
 </tr>
 <td>RNAlater</td>
 <td>Ambion</td>
 <td>AM7021</td>
 <td>&nbsp;</td>
 </tr>
</table>

separation of mouse embryonic facial ectoderm and mesenchyme

Hendiduras orofaciales son los defectos craneofaciales más frecuentes, que afectan a 1,5 de cada 1.000 recién nacidos en todo el mundo 1,2. Hendidura orofacial es causada por el desarrollo facial anormal 3. En, humanos y de ratón de crecimiento inicial y el patrón de la cara se basa en varios brotes pequeños de tejido, las prominencias 4,5 faciales. La cara se deriva de seis pares de prominencias frontales principales: procesos nasales (FNP), prominencias maxilares (MXP) y prominencias mandibulares (MdP). Estas protuberancias consisten en hinchazones de mesénquima que se encajonan en un epitelio suprayacente. Los estudios en múltiples especies han mostrado que la señalización a la diafonía entre el ectodermo y el mesénquima facial es crítica para dar forma a la cara 6. Sin embargo, los detalles mecanicistas sobre los genes implicados en estos relés de señalización son insuficientes. Una forma de obtener una comprensión completa de la expresión génica, factor de transcripción de unión, y las marcas asociadas a la cromatina con el desarrollo de ciertasmesa de ping ectodermo y el mesénquima facial es aislar y caracterizar los compartimentos de tejido separados. Aquí presentamos un método para separar ectodermo y el mesénquima facial en el día embrionario (E) 10,5, una etapa crítica en el desarrollo en la formación de ratón facial que precede a la fusión de las prominencias. Nuestro método es una adaptación del enfoque que hemos utilizado anteriormente para disecar las prominencias faciales 7. En este estudio anterior que había empleado C57BL consanguínea / 6 ratones como esta variedad se ha convertido en un estándar para la genética, la genómica y la morfología facial 8. Aquí, sin embargo, debido a las cantidades más limitadas de tejido disponible, hemos utilizado el outbred cepa CD-1 que es más barato para comprar, más robusto para la cría, y que tiende a producir más embriones (12-18) por camada que cualquier endogámica ratón cepa 8. Después del aislamiento del embrión, proteasa neutra dispasa II se utiliza para tratar el embrión. Entonces, las prominencias faciales fueron diseccionared a cabo, y el ectodermo facial se separó a partir del mesénquima. Este método evita que tanto el ectodermo y el mesénquima facial intacto. Las muestras obtenidas utilizando esta metodología se puede utilizar para técnicas que incluyen la detección de proteínas, la inmunoprecipitación de cromatina (ChIP) de ensayo, los estudios de microarrays, ARN y ss-.

Después Dispasa tratamiento II, el ectodermo del embrión tiende a ser flojo. Las prominencias faciales deben estar intactos después de la disección (Figura 2A). El ectodermo facial aislada es clara y libre de tejido mesénquima (Figura 2B). Para determinar la eficacia del protocolo y para detectar la contaminación cruzada que ensayaron para prosencéfalo expresó ZIC3 9, ectodérmica específico 5 CDH1 y mesenquimales específico SOX10 10 usando la ...

Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Un protocolo para la separación de ectodermo y el mesénquima facial embrión se describe. Utilizamos Dispasa II para el tratamiento de embriones enteros en primer lugar, analizar las prominencias faciales completas, y luego separar el ectodermo facial y mesénquima.

La separación de Ratón Facial ectodermo embrionario y mesénquima

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

separation-of-mouse-embryonic-facial-ectoderm-and-mesenchyme

Research

JoVE Journal

Biology

10.8K vistas.  University of Colorado Denver Anschutz Medical Campus. Un protocolo para la separación de ectodermo y el mesénquima facial embrión se describe. Utilizamos Dispasa II para el tratamiento de embriones enteros en primer lugar, analizar las prominencias faciales completas, y luego separar el ectodermo facial y mesénquima.

Video: Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Separación electroforética de las proteínas

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Investigación

Biología

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 ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

Mediada por adenovirus eliminación de moléculas de señalización genética en cultivos de fibroblastos de embriones de ratón primaria

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Aislamiento y cultivo de células de la zona nefrogénica del riñón de embriones de ratón

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Translocación polarizado de proteínas fluorescentes en Xenopus Ectodermo en respuesta a la señalización de Wnt

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporación de mesénquima craneofacial

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

La diferenciación eficiente de las células madre embrionarias de ratón en neuronas motoras

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparación de las células embrionarias de fibroblastos de ratón adecuados para el cultivo de células estaminales embrionarias humanas pluripotentes inducidas, y

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...